TECHNICAL FIELD
[0001] This invention relates to, the use of bis- and tris-dihydroxyaryl compounds and pharmaceutically
acceptable salts thereof, in the treatment of amyloid diseases, especially Aβ amyloid
disease, such as observed in Alzheimer's disease, IAPP amyloid disease, such as observed
in type 2 diabetes, and synucleinopathies, such as observed in Parkinson's disease,
and in the manufacture of medicaments for such treatment This invention also relates
to the compound 3,4-dihydroxybenzoic acid 3,4-dihydroxyanilide.
BACKGROUND OF THE INVENTION
[0002] Alzheimer's disease is characterized by the accumulation of a 39-43 amino acid peptide
termed the β-amyloid protein or Aβ, in a fibrillar form, existing as extracellular
amyloid plaques and as amyloid within the walls of cerebral blood vessels. Fibrillar
Aβ amyloid deposition in Alzheimer's disease is believed to be detrimental to the
patient and eventually leads to toxicity and neuronal cell death, characteristic hallmarks
of Alzheimer's disease. Accumulating evidence implicates amyloid, and more specifically,
the formation, deposition, accumulation and/or persistence of Aβ fibrils, as a major
causative factor of Alzheimer's disease pathogenesis. In addition, besides Alzheimer's
disease, a number of other amyloid diseases involve formation, deposition, accumulation
and persistence of Aβ fibrils, including Down's syndrome, disorders involving congophilic
angiopathy, such as but not limited to, hereditary cerebral hemorrhage of the Dutch
type, inclusion body myositosis, dementia pugilistica, cerebral β-amyloid angiopathy,
dementia associated with progressive supranuclear palsy, dementia associated with
cortical basal degeneration and mild cognitive impairment.
[0003] Parkinson's disease is another human disorder characterized by the formation, deposition,
accumulation and/or persistence of abnormal fibrillar protein deposits that demonstrate
many of the characteristics of amyloid. In Parkinson's disease, an accumulation of
cytoplasmic Lewy bodies consisting of filaments of α-synuclein/NAC (non-Aβ component)
are believed important in the pathogenesis and as therapeutic targets. New agents
or compounds able to inhibit α-synuclein and/or NAC formation, deposition, accumulation
and/or persistence, or disrupt pre-formed α-synuclein/NAC fibrils (or portions thereof)
are regarded as potential therapeutics for the treatment of Parkinson's and related
synucleinopathies. NAC is a 35 amino acid fragment of α-synuclein that has the ability
to form amyloid-like fibrils either
in vitro or as observed in the brains of patients with Parkinson's disease. The NAC fragment
of α-synuclein is a relative important therapeutic target as this portion of α-synuclein
is believed crucial for formation of Lewy bodies as observed in all patients with
Parkinson's disease, synucleinopathies and related disorders.
[0004] A variety of other human diseases also demonstrate amyloid deposition and usually
involve systemic organs (i.e. organs or tissues lying outside the central nervous
system), with the amyloid accumulation leading to organ dysfunction or failure. These
amyloid diseases (discussed below) leading to marked amyloid accumulation in a number
of different organs and tissues, are known as systemic amyloidoses. In other amyloid
diseases, single organs may be affected such as the pancreas in 90% of patients with
type 2 diabetes. In this type of amyloid disease, the beta-cells in the islets of
Langerhans in pancreas are believed to be destroyed by the accumulation of fibrillar
amyloid deposits consisting primarily of a protein known as islet amyloid polypeptide
(IAPP). Inhibiting or reducing such IAPP amyloid fibril formation, deposition, accumulation
and persistence is believed to lead to new effective treatments for type 2 diabetes.
In Alzheimer's disease, Parkinson's and "systemic" amyloid diseases, there is currently
no cure or effective treatment, and the patient usually dies within 3 to 10 years
from disease onset.
[0005] The amyloid diseases (amyloidoses) are classified according to the type of amyloid
protein present as well as the underlying disease. Amyloid diseases have a number
of common characteristics including each amyloid consisting of a unique type of amyloid
protein. The amyloid diseases include, but are not limited to, the amyloid associated
with Alzheimer's disease, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis
of the Dutch type, dementia pugilistica, inclusion body myositosis (
Askanas et al, Ann. Neurol. 43:521-560, 1993) and mild cognitive impairment (where the specific amyloid is referred to as beta-amyloid
protein or Aβ), the amyloid associated with chronic inflammation, various forms of
malignancy and Familial Mediterranean Fever (where the specific amyloid is referred
to as AA amyloid or inflammation-associated amyloidosis), the amyloid associated with
multiple myeloma and other B-cell dyscrasias (where the specific amyloid is referred
to as AL amyloid), the amyloid associated with type 2 diabetes (where the specific
amyloid protein is referred to as amylin or islet amyloid polypeptide or IAPP), the
amyloid associated with the prion diseases including Creutzfeldt-Jakob disease, Gerstmann-Straussler
syndrome, kuru and animal scrapie (where the specific amyloid is referred to as PrP
amyloid), the amyloid associated with long-term hemodialysis and carpal tunnel syndrome
(where the specific amyloid is referred to as α
2-microglobulin amyloid), the amyloid associated with senile cardiac amyloidosis and
Familial Amyloidotic Polyneuropathy (where the specific amyloid is referred to as
transthyretin or prealbumin), and the amyloid associated with endocrine tumors such
as medullary carcinoma of the thyroid (where the specific amyloid is referred to as
variants of procalcitonin). In addition, the α-synuclein protein which forms amyloid-like
fibrils, and is Congo red and Thioflavin S positive (specific stains used to detect
amyloid fibrillar deposits), is found as part of Lewy bodies in the brains of patients
with Parkinson's disease, Lewy body disease (
Lewy in Handbuch der Neurologie, M. Lewandowski, ed., Springer, Berlin pp. 920-933,
1912;
Pollanen et al, J. Neuropath. Exp. Neurol. 52:183-191, 1993;
Spillantini et al, Proc. Natl. Acad. Sci. USA-95:6469-6473, 1998;
Arai et al, Neurosci. Lett. 259:83-86, 1999), multiple system atrophy (
Wakabayashi et al, Acta Neuropath. 96:445-452, 1998), dementia with Lewy bodies, and the Lewy body variant of Alzheimer's disease. For
purposes of this disclosure, Parkinson's disease, due to the fact that fibrils develop
in the brains of patients with this disease (which are Congo red and Thioflavin S
positive, and which contain predominant beta-pleated sheet secondary structure), is
now regarded as a disease that also displays the characteristics of an amyloid-like
disease.
[0006] Systemic amyloidoses which include the amyloid associated with chronic inflammation,
various forms of malignancy and familial Mediterranean fever (i.e. AA amyloid or inflammation-associated
amyloidosis) (
Benson and Cohen, Arth. Rheum. 22:36-42, 1979;
Kamei et al, Acta Path. Jpn. 32:123-133, 1982;
McAdam et aL, Lancet 2:572-573, 1975;
Metaxas, Kidney Int. 20:676-685, 1981), and the amyloid associated with multiple myeloma and other B-cell dyscrasias (i.e.
AL amyloid) (
Harada et al., J. Histochem. Cytochem. 19:1-15, 1971), as examples, are known to involve amyloid deposition in a variety of different
organs and tissues generally lying outside the central nervous system. Amyloid deposition
in these diseases may occur, for example, in liver, heart, spleen, gastrointestinal
tract, kidney, skin, and/or lungs
Johnson et al, N. Engl. J. Med. 321:513-518,1989). For most of these amyloidoses, there is no apparent cure or effective treatment
and the consequences of amyloid deposition can be detrimental to the patient For example,
amyloid deposition in the kidney may lead to renal failure, whereas amyloid deposition
in the heart may lead to heart failure. For these patients, amyloid accumulation in
systemic organs leads to eventual death generally within 3-5 years. Other amyloidoses
may affect a single organ or tissue such as observed with the Aβ amyloid deposits
found in the brains of patients with Alzheimer's disease and Down's syndrome: the
PrP amyloid deposits found in the brains of patients with Creutzfeldt-Jakob disease,
Gerstmann-Straussler syndrome, and kuru; the islet amyloid (IAPP) deposits found in
the islets of Langerhans in the pancreas of 90% of patients with type 2 diabetes (
Johnson et al, N. Engl. J. Med. 321:513-518, 1989;
Lab. Invest. 66:522 535, 1992); the α
2-microglobulin amyloid deposits in the medial nerve leading to carpal tunnel syndrome
as observed in patients undergoing long-term hemodialysis (
Geyjo et al, Biochem. Biophys. Res. Comm. 129:701-706, 1985;
Kidney Int. 30:385-390, 1986); the prealbumin/ transthyretin amyloid observed in the hearts of patients with senile
cardiac amyloid; and the prealbumin/transthyretin amyloid observed in peripheral nerves
of patients who have familial amyloidotic polyneuropathy (
Skinner and Cohen, Biochem. Biophys. Res. Comm. 99:1326-1332, 1981;
Saraiva et al, J. Lab. Clin. Med. 102:590-603, 1983;
J. Clin. Invest. 74:104-119, 1984;
Tawara et al, J. Lab. Clin. Med 98:811-822, 1989).
[0007] Alzheimer's disease also puts a heavy economic burden on society. A recent study
estimated that the cost of caring for one Alzheimer's disease patient with severe
cognitive impairments at home or in a nursing home, is more than $47,000 per year
(
A Guide to Understanding Alzheimer's Disease and Related Disorders). For a disease that can span from 2 to 20 years, the overall cost of Alzheimer's
disease to families and to society is staggering. The annual economic toll of Alzheimer's
disease in the United States in terms of health care expenses and lost wages of both
patients and their caregivers is estimated at $80 to $100 billion (
2003 Progress Report on Alzheimer's Disease).
[0008] Tacrine hydrochloride ("Cognex"), the first FDA approved drug for Alzheimer's disease,
is a acetylcholinesterase inhibitor (
Cutler and Sramek, N. Engl. J. Med. 328:808 810, 1993). However, this drug has showed limited success in producing cognitive improvement
in Alzheimer's disease patients and initially had major side effects such as liver
toxicity. The second FDA approved drug, donepezil ("Aricept"), which is also an acetylcholinesterase
inhibitor, is more effective than tacrine, by demonstrating slight cognitive improvement
in Alzheimer's disease patients (
Barner and Gray, Ann. Pharmacotherapy 32:70-77, 1998;
Rogers and Friedhoff, Eur. Neuropsych. 8:67-75, 1998), but is not believed to be a cure. Therefore, it is clear that there is a need for
more effective treatments for Alzheimer's disease patients.
Amyloid as a therapeutic target for Alzheimer's disease
[0009] Alzheimer's disease is characterized by the deposition and accumulation of a 39-43
amino acid peptide termed the beta-amyloid protein, Aβ or β/A4 (
Glenner and Wong, Biochem. Biophys. Res. Comm. 120:885-890,1984;
Masters et al., Proc. Natl. Acad. Sci. USA 82:4245-4249, 1985;
Husby et aL, Bull. WHO 71:105-108, 1993). Aβ is derived by protease cleavage from larger precursor proteins termed β-amyloid
precursor proteins (APPs) of which there are several alternatively spliced variants.
The most abundant forms of the APPs include proteins consisting of 695, 751 and 770
amino acids (
Tanzi et aL, Nature 31:528-530, 1988).
[0010] The small Aβ peptide is a major component that makes up the amyloid deposits of "plaques"
in the brains of patients with Alzheimer's disease. In addition, Alzheimer's disease
is characterized by the presence of numerous neurofibrillary "tangles", consisting
of paired helical filaments which abnormally accumulate in the neuronal cytoplasm
(
Grundke-Iqbal et aL, Proc. Natl. Acad Sci. USA 83:4913-4917, 1986;
Kosik et aL, Proc. Natl. Acad. Sci. USA 83:4044-4048, 1986;
Lee et al., Science 251:675-678, 1991). The pathological hallmark of Alzheimer's disease is therefore the presence of "plaques"
and "tangles", with amyloid being deposited in the central core of the plaques. The
other major type of lesion found in the Alzheimer's disease brain is the accumulation
of amyloid in the walls of blood vessels, both within the brain parenchyma and in
the walls of meningeal vessels that lie outside the brain. The amyloid deposits localized
to the walls of blood vessels are referred to as cerebrovascular amyloid or congophilic
angiopathy (
Mandybur, J. Neuropath. Exp. Neurol. 45:79-90, 1986;
Pardridge et al., J. Neurochem. 49:1394-1401, 1987)
[0011] For many years there has been an ongoing scientific debate as to the importance of
"amyloid" in Alzheimer's disease, and whether the "plaques" and "tangles" characteristic
of this disease were a cause or merely a consequence of the disease. Within the last
few years, studies now indicate that amyloid is indeed a causative factor for Alzheimer's
disease and should not be regarded as merely an innocent bystander. The Alzheimer's
Aβ protein in cell culture has been shown to cause degeneration of nerve cells within
short periods of time (
Pike et al., Br. Res. 563:311-314, 1991;
J. Neurochem. 64:253-265, 1995). Studies suggest that it is the fibrillar structure (consisting of a predominant
β-pleated sheet secondary structure), characteristic of all amyloids, that is responsible
for the neurotoxic effects. Aβ has also been found to be neurotoxic in slice cultures
of hippocampus (
Harrigan et al., Neurobiol. Aging 16:779-789, 1995) and induces nerve cell death in transgenic mice (
Games et al., Nature 373:523-527, 1995;
Hsiao et al., Science 274:99-102, 1996). Injection of the Alzheimer's Aβ into rat brain also causes memory impairment and
neuronal dysfunction (
Flood et aL, Proc. Natl. Acad. Sci. USA 88:3363-3366, 1991;
Br. Res. 663:271-276, 1994).
[0012] Probably, the most convincing evidence that Aβ amyloid is directly involved in the
pathogenesis of Alzheimer's disease comes from genetic studies. It was discovered
that the production of Aβ can result from mutations in the gene encoding, its precursor,
β-amyloid precursor protein (Van
Broeckhoven et al., Science 248:1120-1122, 1990;
Murrell et al., Science 254:97-99, 1991;
Haass et al., Nature Med. 1:1291-1296, 1995). The identification of mutations in the beta-amyloid precursor protein gene that
cause early onset familial Alzheimer's disease is the strongest argument that amyloid
is central to the pathogenetic process underlying this disease. Four reported disease-causing
mutations have been discovered Which demonstrate the importance of Aβ in causing familial
Alzheimer's disease (reviewed in
Hardy, Nature Genet. 1:233-234, 1992). All of these studies suggest that providing a drug to reduce, eliminate or prevent
fibrillar Aβ formation, deposition, accumulation and/or persistence in the brains
of human patients will serve as an effective therapeutic.
Parkinson's Disease and Synucleinopathies
[0013] Parkinson's disease is a neurodegenerative disorder that is pathologically characterized
by the presence of intracytoplasmic Lewy bodies (
Lewy in Handbuch der Neurologie, M. Lewandowski, ed., Springer, Berlin, pp. 920-933,
1912;
Pollanen et aL, J. Neuropath. Exp. Neurol. 52:183-191, 1993), the major components of which are filaments consisting of α-synuclein (
Spillantini et aL, Proc. Natl. Acad. Sci. USA 95:6469-6473, 1998;
Arai et al., Neurosci. Lett. 259:83-86, 1999), an 140-amino acid protein (
Ueda et al., Proc. Natl. Acad. Sci. USA 90:11282-11286, 1993). Two dominant mutations in α-synuclein causing familial early onset Parkinson's
disease have been described suggesting that Lewy bodies contribute mechanistically
to the degeneration of neurons in Parkinson's disease and related disorders (
Polymeropoulos et al., Science 276:2045-2047, 1997;
Kruger et al., Nature Genet. 18:106-108, 1998). Recently,
in vitro studies have demonstrated that recombinant α-synuclein can indeed form Lewy body-like
fibrils (
Conway et al., Nature Med. 4:1318-1320, 1998;
Hashimoto et al., Brain Res. 799:301-306,1998;
Nahri et al., J. Biol Chem. 274:9843-9846, 1999). Most importantly, both Parkinson's disease-linked α-synuclein mutations accelerate
this aggregation process, demonstrating that such
in vitro studies may have relevance for Parkinson's disease pathogenesis. Alpha-synuclein
aggregation and fibril formation fulfills of the criteria of a nucleation-dependent
polymerization process (
Wood et aL, J. Biol. Chem. 274:19509-19512, 1999). In this regard α-synuclein fibril formation resembles that of Alzheimer's β-amyloid
protein (Aβ) fibrils. Alpha-synuclein recombinant protein, and non-Aβ component (known
as NAC), which is a 35-amino acid peptide fragment of α-synuclein, both have the ability
to form fibrils when incubated at 37°C, and are positive with amyloid stains such
as Congo red (demonstrating a red/green birefringence when viewed under polarized
light) and Thioflavin S (demonstrating positive fluorescence) (
Hashimoto et al., Brain Res. 799:301-306, 1998;
Ueda et aL, Proc. Natl. Acad. Sci. USA 90:11282-11286, 1993).
[0014] Synucleins are a family of small, presynaptic neuronal proteins composed of α-, β-,
and γ-synucleins, ofwhich only α-synuclein aggregates have been associated with several
neurological diseases (
Ian et al., Clinical Neurosc. Res. 1:445-455, 2001;
Trojanowski and Lee, Neurotoxicology 23:457-460, 2002). The role of synucleins (and in particular, alpha-synuclein) in the etiology of
a number of neurodegenerative and/or amyloid diseases has developed from several observations.
Pathologically, synuclein was identified as a major component of Lewy bodies, the
hallmark inclusions of Parkinson's disease, and a fragment thereof was isolated from
amyloid plaques of a different neurological disease, Alzheimer's disease. Biochemically,
recombinant α-synuvlein was shown to form amyloid-like fibrils that recapitulated
the ultrastructural features of alpha-synuclein isolated from patients with dementia
with Lewy bodies, Parkinson's disease and multiple system atrophy. Additionally, the
identification of mutations within the synuclein gene, albeit in rare cases of familial
Parkinson's disease, demonstrated an unequivocal link between synuclein pathology
and neurodegenerative diseases. The common involvement of α-synuclein in a spectrum
of diseases such as Parkinson's disease, dementia with Lewy bodies, multiple system
atrophy and the Lewy body variant of Alzheimer's disease has led to the classification
of these diseases under the umbrella term of "synucleinopathies".
[0015] Parkinson's disease α-synuclein fibrils, like the Aβ fibrils of Alzheimer's disease,
also consist of a predominantly β-pleated sheet structure. Therefore, compounds found
to inhibit Alzheimer's disease Aβ amyloid fibril formation are also anticipated to
be effective in the inhibition of α-synuclein/ NAC fibril formation, as shown from
Examples in the present invention. These compounds would therefore also serve as therapeutics
for Parkinson's disease and other synucleinopathies, in addition to having efficacy
as a therapeutic for Alzheimer's disease, type 2 diabetes, and other amyloid disorders.
[0016] Discovery and identification of new compounds or agents as potential therapeutics
to arrest amyloid formation, deposition, accumulation and/or persistence that occurs
in Alzheimer's disease, Parkinson's disease, type II diabetes, and other amyloidoses
are desperately sought
SUMMARY OF THE INVENTION
[0017] In a first aspect, this invention relates to bis- and tris-dihydroxyaryl compounds
and pharmaceutically acceptable salts thereof for use in the treatment of amyloid
diseases and synucleinopathies.
[0018] The compounds that are useful in treating an amyloid disease or synucleinopathy are:
compounds of the formula:
where:
R is a C2-C10 alkylene group, in which there is optionally 1 double bonds; 1 or 2 non-adjacent
ethylene groups are replaced by C(O)NR1, or NR1C(O)-, where R1 is H, or lower alkyl.
[0019] The invention also relates to the compound 3,4-dihydroxybenzoic acid 3,4-dihydroxy-anilide
(compound 51).
[0022] EP 0,324,521 discloses compounds of the general formula (I):
[0023] These compounds can be used to treat hematologic diseases in warm-blooded living
beings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024]
Figure 1 is four circular dichroism spectra showing examples of Alzheimer's disease
Aβ fibril disruption by compounds 4, 12, 51 and 61.
Figure 2 is a circular dichroism spectrum showing an example of Alzheimer's disease
Aβ fibril disruption by compound 78.
Figure 3 is three circular dichroism spectra showing examples of Alzheimer's disease
Aβ fibril disruption (in a dose-dependent manner) by compounds 12, 51 and 61.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0025] In this application, the following terms shall have the following meanings, without
regard to whether the terms are used vatiantly elsewhere in the literature or otherwise
in the known art
[0026] The compounds are referred to generally as bis- and tris-dihydroxyaryl compounds,
or sometimes just as "dihydroxyaryl compounds". It will be noted that compound #84
has an additional hydroxy group, but does have two dihydroxyaryl groups; while compound
#86 has only one dihydroxyaryl group but has an additional phenolic hydroxyl moiety.
[0027] "Methylenedioxy analogs" refers to the compounds in which each of the pairs of adjacent
hydroxyl moieties of the dihydroxyaryl groups have been replaced by methylenedioxy
groups. The methylenedioxy compounds are illustrated and referred to as compounds
#1B to #86B or DC-0001B to DC-0086B. The methylenedioxy groups also are convenient
intermediate protecting groups for the dihydroxy moieties and therefore these disclosed
compounds are believed to also serve as effective prodrugs. The methylenedioxy analogs
#1B to #80B are illustrated in Example 30.
[0028] "Pharmaceutically acceptable esters" refers to the compounds where the hydroxyl moieties
of the dihydroxyaryl groups of the compounds are esterified with an acid or acids
that result in a pharmaceutically acceptable poly(ester). The compounds are shown
in Example 31 as acetylated, and these acetylated compounds are illustrated and referred
to as compounds #1C to #86C or DC-0001C to DC-0086C;
[0029] The ester groups are expected to serve as intermediate protecting groups for the
hydroxyl moieties and therefore the pharmaceutically acceptable esters are expected
to serve as effective prodrugs for their underlying bis- and tris-dihydroxyaryl compounds.
[0030] Chemical structures for each of the compounds of this invention (with the note that
the acetates are shown as representative of the pharmaceutically acceptable esters
as a class) are shown. The names of the compounds are variously IUPAC names [names
derived according to the accepted IUPAC (International Union of Pure and Applied Chemistry)
system established by the coalition of the Commission on Nomenclature of Organic Chemistry
and the Commission on Physical Organic Chemistry, as can be found at
http://www.chem.qmul.ac.uk/iupac], names derived from IUPAC names by addition or substitution (for example, by the
use of "3,4-methylenedioxyphenyl" derived from "phenyl" instead of "benzo[1,3]dioxol-5-yl"),
and names derived from the names of reactants (for example, by the use of "3,4-dihydroxybenzoic
acid 3,4-dihydroxyanilide" instead of "N-(3,4-dihydroxy-phenyl)-3,4-dihydroxybenzamide").
However, the names used are explicitly equated to chemical structures, and are believed
to be readily understood by a person of ordinary skill in the art.
[0031] "Mammal" includes both humans and non-human mammals, such as companion animals (cats,
dogs, and the like), laboratory animals (such as mice, rats, guinea pigs, and the
like) and farm animals (cattle, horses, sheep, goats, swine, and the like).
[0032] "Pharmaceutically acceptable excipient" means an excipient that is conventionally
useful in preparing a pharmaceutical composition that is generally safe, non-toxic,
and desirable, and includes excipients that are acceptable for veterinary use as well
as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid,
or, in the case of an aerosol composition, gaseous.
[0033] "Pharmaceutically acceptable salt" means a salt that is pharmaceutically acceptable
and have the desired pharmacological properties. Such salts include salts that may
be formed where acidic protons present in the compounds are capable of reacting with
inorganic or organic bases. Suitable inorganic salts include those formed with the
alkali metals, e.g. sodium and potassium, magnesium, calcium, and aluminum. Suitable
organic salts include those formed with organic bases such as the amine bases, e.g.
ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and
the like. Such salts also include acid addition salts formed with inorganic acids
(e.g. hydrochloric and hydrobromic acids) and organic acids (e.g. acetic acid, citric
acid, maleic acid, and the alkane- and arene-sulfonic acids such as methanesulfonic
acid and benzenesulfonic acid). When there are two acidic groups present, a pharmaceutically
acceptable salt may be a mono-acid-mono-salt or a di-salt; and similarly where there
are more than two acidic groups present, some or all of such groups can be salified.
[0034] A "therapeutically effective amount" in general means the amount that, when administered
to a subject or animal for treating a disease, is sufficient to affect the desired
degree of treatment for the disease. A "therapeutically effective amount" or a "therapeutically
effective dosage" preferably inhibits, reduces, disrupts, disassembles amyloid or
synuclein fibril formation, deposition, accumulation and/or persistence, or treats
a disease associated with these conditions, such as an amyloid disease or a synucleinopathy,
by at least 20%, more preferably by at least 40%, even more preferably by at least
60%, and still more preferably by at least 80%, relative to an untreated subject Effective
amounts of a compound of this invention or composition thereof for treatment of a
mammalian subject are about 0.1 to about 1000 mg/Kg of body weight of the subject/day,
such as from about 1 to about 100 mg/Kg/day, especially from about 10 to about 100
mg/Kg/day. A broad range of disclosed composition dosages are believed to be both
safe and effective.
[0035] "Treating" or "treatment" of a disease includes preventing the disease from occurring
in a mammal that may be predisposed to the disease but does not yet experience or
exhibit symptoms of the disease (prophylactic treatment), inhibiting the disease (slowing
or arresting its development), providing relief from the symptoms or side-effects
of the disease (including palliative treatment), and relieving the disease (causing
regression of the disease), such as by disruption of pre-formed amyloid or synuclein
fibrils. One such preventive treatment may be use of the disclosed compounds for the
treatment of Mild Cognitive impairment (MCI).
[0036] "NAC" (non-Aβ component) is a 35-amino acid peptide fragment of α-synuclein, which
like α-synuclein, has the ability to form amyloid-like fibrils when incubated at 37°C,
and is positive with amyloid stains such as Congo red (demonstrating a red/green birefringence
when viewed under polarized light) and Thioflavin S (demonstrating positive fluorescence)
(
Hashimoto et aL, Brain Res. 799:301-306, 1998;
Ueda et al., Proc. Natl. Acad. Sci. U.S.A. 90:11282-11286,1993). Inhibition of NAC fibril formation, deposition, accumulation, aggregation, and/or
persistence is believed to be effective treatment for a number of diseases involving
α-synuclein, such as Parkinson's disease, Lewy body disease and multiple system atrophy.
[0037] "Fibrillogenesis" refers to the formation, deposition, accumulation and/or persistence
of amyloid fibrils, filaments, inclusions, deposits, as well as synuclein (usually
involving α-synuclein) and/or NAC fibrils, filaments, inclusions, deposits or the
like.
[0038] "Inhibition of fibrillogenesis" refers to the inhibition of formation, deposition,
accumulation and/or persistence of such amyloid fibrils or synuclein fibril-like deposits.
[0039] "Disruption of fibrils or fibrillogenesis" refers to the disruption of pre-formed
amyloid or synuclein fibrils, that usually exist in a pre-dominant β-pleated sheet
secondary structure. Such disruption by compounds of the invention may involve marked
reduction or disassembly of amyloid or synuclein fibrils as assessed by various methods
such as circular dichroism spectroscopy, Thioflavin T fluorometry, Congo red binding,
SDS-PAGE/Western blotting, as demonstrated by the Examples presented in this application.
[0040] "A pharmaceutical agent" or "pharmacological agent" or "pharmaceutical composition"
refers to a compound or combination of compounds used for treatment, preferably in
a pure or near pure form. In the specification, pharmaceutical or pharmacological
agents include the compounds of this invention. The compounds are desirably purified
to 80% homogeneity, and preferably to 90% homogeneity. Compounds and compositions
purified to 99.9% homogeneity are believed to be advantageous. As a test or confirmation,
a suitable homogeneous compound on HPLC would yield, what those skilled in the art
would identify as a single sharp-peak band.
Compounds
[0041] The compound of this invention is 3,4-dihydroxy benzoic acid 3,4-dihydroxyanilide.
Disclosed herein are:
- (1) compounds of the formula:
where:
R is a C1-C10 alkylene group, in which, when the number of carbon atoms is at least 2, there are
optionally 1 or 2 non-adjacent double bonds; 1 to 3 non-adjacent methylene groups
are optionally replaced by NR' (where R' is H, alkyl, or acyl), O, or S; and 1 or
2 methylene groups are optionally replaced by a carbonyl or hydroxymethylene group;
and
- (2) the compounds that are:
3,4,3',4'-tetrahydroxybenzoin; 3,4,3',4'-tetrahydroxydesoxybenzoin; 3,4,3',4'-tetrahydroxydiphenylmethane;
1,2-bis(3,4-dihydroxyphenyl)ethane; 1,3-bis(3,4-dihydroxyphenyl)propane; 3,4,3',4'-tetrahydroxychalcone;
3,5-bis(3,4-dihydroxyphenyl)-1-methyl-2-pyrazoline; 4,6-bis(3,4-dihydroxyphenyl)-3-cyano-2-methylpyridine;1,4-bis(3,4-dihydroxybenzyl)piperazine;
N,N'-bis(3,4-dihydroxybenzyl)-N,N'-dimethyl-ethylenediamine; 2,5-bis(3,4-dihydroxybenzyl)-2,5-diaza[2.2.1]bicycloheptane;
N,N'-bis(3,4-dihydroxybenzyl)-trans-1,2-diaminocyclohexane; N,N'-bis(3,4-dihydroxybenzyl)-trans-1,4-diaminocyclohexane; N,N'-bis(3,4-dihydroxybenzyl)-cis-1,3-bis(aminomethyl)cyclohexane; N-(3,4-dihydroxybenzyl)proline 3,4-dihydroxybenzylamide;
2-(3,4-dihydroxybenzyl)isoquinoline-3-carboxylic acid 3,4-dihydroxyphenethylamide;
2,6-bis(3,4-dihydroxybenzyl)cyclohexanone; 3,5-bis(3,4-dihydroxybenzyl)-1-methyl-4-piperidinone;
2,4-bis(3,4-dihydroxybenzyl)-3-tropinone; tris(3,4-dihydroxybenzyl)methane; α-(3,4-dihydroxybenzamido)-3,4-dihydroxycinnamic
acid 3,4-dihydroxybenzyl amide; 4-(3,4-dihydroxy-benzylaminomethylene)-2-(3,4-dihydroxyphenyl)oxazolin-5-one;
1,4-bis(3,4-dihydroyxybenzoyl)piperazine; N,N'-bis(3,4-dihydroxybenzoyl)-N,N'-dimethylethylenediamine;
2,5-bis(3,4-dihydroxybenzoyl)-2,5-diaza[2.2.1]bicycloheptane; N,N'-bis(3,4-dihydroxybenzoyl)-trans-1,2-diaminocyclohexane; N,N'-bis(3,4-dihydroxybenzoyl)-cis-1,3-bis(aminomethyl)cyclohexane; 3,6-bis(3,4-dihydroxybenzyl)-2,5-diketopiperazine;
3,6-bis(3,4-dihydroxybenzylidene)-1,4-dimethyl-2,5-diketopiperazine; N-(3,4-dihydroxyphenylacetyl)proline-3,4-dihydroxyanilide;
2,3-bis(3,4-dihydroxyphenyl)butane; 1,3-bis(3,4-dihydroxybenzyl)benzene;1,4-bis(3,4-dihydroxybenzyl)benzene;2,6-bis(3,4-dihydroxybenzyl)-pyridine;
2,5-bis(3,4-dihydroxybenzyl)thiophene; 2,3-bis(3,4-dihydroxybenzyl)thiophene; 1,2-bis(3,4-dihydroxyphenyl)cyclohexane;1,4-bis(3,4-dihydroxyphenyl)cyclohexane;
3,7-bis(3,4-dihydroxyphenyl)bicyclo[3.3.0]octane; 2,3-bis(3,4-dihydroxyphenyl)-1,7,7-trimethyl-bicyclo[2.2.1]heptane;
1,2-bis(3,4-dihydroxyphenoxy)ethane; 1,3-bis(3,4-dihydroxyphenoxy)propane; trans-1,2-bis(3,4-dihydroxy-phenoxy)cyclopentane; N-(3,4-dihydroxybenzyl)-3-(3,4-dihydroxyphenoxy)-2-hydroxypropylamine;
3,4-dihydroxyphenoxyacetic acid 3,4-dihydroxyanilide; 3,4-dihydroxyphenoxyacetic acid
3,4-dihydroxy benzylamide; 3,4-dihydroxyphenoxyacetic acid 3,4-dihydroxyphenethylamide;
3,4-dihydroxybenzoic acid p-(3,4-dihydroxyphenoxy)anilide; 3,4-dihydroxybenzoic acid
o-(3,4-dihydroxyphenoxy)anilide; 2,6-bis (3,4-dihydroxyphenoxy)pyridine; 3,4-dihydroxy-benzoic
acid 3,4-dihydroxybenzylamide; 3,4-dihydroxybenzoic acid 3,4-dihydroxyphenethylamide;
3,4-dihydroxyphenyl acetic acid 3,4-dihydroxyanilide; 3,4-dihydroxyphenylacetic acid
3,4-dihydroxybenzyl-amide; 3,4-dihydroxyphenylacetic acid 3,4-dihydroxyphenethylamide;
3-(3,4-dihydroxyphenyl)propionic acid 3,4-dihydroxyanilide; 3-(3,4-dihydroxyphenyl)
propionic acid 3,4-dihydroxybenzylamide; 3-(3,4-dihydroxyphenyl)propionic acid 3,4-dihydroxyphenethylamide;
3,4-dihydroxycinnamic acid 3,4-dihydroxyanilide; 3,4-dihydroxycinnamic acid 3,4-dihydroxybenzylamide;
3,4-dihydroxycinnamic acid 3,4-dihydroxyphenethylamide; oxalic acid bis(3,4-dihydroxyanilide);
oxalic acid bis(3,4-dihydroxybenzylamide); oxalic acid bis(3,4-dihydroxyphenethylamide);
succinic acid bis(3,4-dihydroxyanilide); succinic acid bis(3,4-dihydroxybenzylamide);
succinic acid bis(3,4-dihydroxyphenethylamide); maleic acid bis(3,4-dihydroxyanilide);
maleic acid bis(3,4-dihydroxybenzylamide); fumaric acid bis(3,4-dihydroxy-anilide);
fumaric acid bis(3,4-dihydroxybenzylamide); bis(3,4-dihydroxybenzyl)amine; N-(3,4-dihydroxy-benzyl)-3,4-dihydroxyphenethylamine;
tris(3,4-dihydroxybenzyl)amine; 1,3-bis(3,4-dihydroxyphenyl)urea; 1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxybenzyl)urea,
1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxy-phenethyl)urea; 3-deoxy-3-(3,4-dihydroxybenzyl)aminoepicatechin;
3-deoxy-3-(3,4-dihydroxyphenethyl)-aminoepicatechin; 2,3,6,7-tetrahydroxy-9,10-epoxy-9,10-dihydroacridine;
10-aminoanthracene-1,2,7,8-tetraol; acridine-1,2,6,7-tetraol; phenoxazine-2,3,7,8,10-pentaol;
dibenzo[c,f][2,7]napthyridine-2,3,10,11-tetraol; and 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-2,10,11-triol;
- (3) the methylenedioxy analogs and pharmaceutically acceptable esters of the compounds
of (1) and (2); and
- (4) the pharmaceutically acceptable salts of the compounds of (1) to (3).
[0042] Within the compounds disclosed above a first group of compounds is the compounds
selected from the group consisting of:
- (1) compounds of the formula:
where:
R is a C1-C10, especially a C1-6, alkylene group, in which, when the number of carbon atoms is at least 2, there are
optionally 1 or 2 non-adjacent double bonds; 1 to 3 non-adjacent methylene groups
are optionally replaced by NR' (where R' is H, C1-3 alkyl, or C2-4 acyl), O, or S, especially NH or N-CH3; and 1 or 2 methylene groups are optionally replaced by a carbonyl or hydroxymethylene
group;
- (2) the methylenedioxy analogs and pharmaceutically acceptable tetraesters thereof;
and
- (3) the pharmaceutically acceptable salts of the compounds of (1) and (2).
[0043] Within this first group, a subgroup of compounds is the group of compounds selected
from the group consisting of:
- (1) compounds of the formula:
where:
R is a C2-C10, especially a C2-6, alkylene group, in which there is optionally 1 double bond; and 1 or 2 non-adjacent
ethylene groups are replaced by -C(O)NR'- or - NR'C(O)- (where R' is H or lower alkyl);
- (2) the methylenedioxy analogs and pharmaceutically acceptable tetraesters thereof;
and
- (3) the pharmaceutically acceptable salts of compounds of (1) and (2).
[0044] Within the compounds of this invention, a second group of compounds is:
- (1) the compounds that are:
3,4,3',4'-tetrahydroxybenzoin; 3,4,3',4'-tetrahydroxydesoxybenzoin; 3,4,3',4'-tetrahydroxydiphenylmethane;
1,2-bis(3,4-dihydroxyphenyl)ethane; 1,3-bis(3,4-dihydroxyphenyl)propane; 3,4,3',4'-tetrahydroxychalcone;
3,5-bis(3,4-dihydroxyphenyl)-1-methyl-2-pyrazoline;4,6-bis(3,4-dihydroxyphenyl)-3-cyano-2-methylpyridine;
1,4-bis(3,4-dihydroxybenzyl)piperazine; N,N'-bis(3,4-dihydroxybenzyl)-N,N'-dimethylethylenediamine;
2,5-bis(3,4-dihydroxybenzyl)-2,5-diaza[2.2.1]bicycloheptane; N,N'-bis(3,4-dihydroxy-benzyl)-trans-1,2-diaminocyclohexane; N,N'-bis(3,4-dihydroxybenzyl)-trans-1,4-diaminocyclohexane; N,N'-bis(3,4-dihydroxybenzyl)-cis-1,3-bis(aminomethyl)cyclohexane; N-(3,4-dihydroxybenzyl)proline 3,9-dihydroxybenzylamide;
2-(3,4-dihydroxoxybenzyl)isoquinoline-3-carboxylic acid 3,4-dihydroxy-phenethylamide;
2,6-bis(3,4-dihydroxybenzyl)cyclohexanone; 3,5-bis(3,4-,dihydroxybenzyl)-1-methyl-4-piperidinone;
2,4-bis(3,4-dihydroxybenzyl)-3-tropinone; tris(3,4-dihydroxybenzyl)methane; a-(3,4dihydroxybenzamido)-3,4-dihydroxycinnamic
acid 3,4-dihydroxybenzyl amide; 4-(3,4-dihydroxy-benzylaminomethylene)-2-(3,4-dihydroxyphenyl)oxazolin-5-one;
1,4-bis(3,4-dihydroxybenzoyl)piperazine; N,N'-bis(3,4-dihydroxybenzoyl)-N,N'-dimethylethylenediamine;
2,5-bis(3,4-dihydroxybenzoyl)-2,5-diaza[2.2.1]bicycloheptane; N,N'-bis(3,4-dihydroxybenzoyl)-trans-1,2-diaminocyclohexane; N,N'-bis(3,4-dihydroxybenzoyl)-cis-1,3-bis(aminomethyl)cyclohexane; 3,6-bis(3,4-dihydroxybenzyl)-2,5-diketopiperazine;
3,6-bis(3,4-dihydroxybenzylidene)-1,4-dimethyl-2,5-diketopiperazine; N-(3,4-dihydroxyphenylacetyl)proline-3,4-dihydroxyanilide;
2,3-bis(3,4-dihydroxyphenyl)butane; 1,3-bis(3,4-dihydroxybenzyl)benzene; 1,4-bis(3,4-dihydroxybenzyl)benzene;
2,6-bis(3,4-dihydroxybenzyl)-pyridine; 2,5-bis(3,4-dihydroxybenzyl)thiophene; 2,3-bis(3,4-dihydroxybenzyl)thiophene;
1,2-bis(3,4-dihydroxyphenyl)cyclohexane;1,4-bis(3,4-dihydroxyphenyl)cyclohexane; 3,7-bis(3,4-dihydroxy-phenyl)bicyclo[3.3.0]octane;
2,3-bis(3,4-dihydroxyphenyl)-1,7,7-trimethyl-bicyclo[2.2.1]heptane; 1,2-bis(3,4-dihydroxyphenoxy)ethane;1,3-bis(3,4-dihydroxyphenoxy)propane;
trans-1,2-bis(3,4-dihydroxy-phenoxy)cyclopentane; N-(3,4-dihydroxybenzyl)-3-(3,4-dihydroxyphenoxy)-2-hydroxypropylamine;
3,4-dihydroxyphenoxyacetic acid 3,4-dihydroxyanilide; 3,4-dihydroxyphenoxyacetic acid
3,4-dihydroxy-benzylamide; 3,4-dihydroxyphenoxyacetic acid 3,4-dihydroxyphenethylamide;
3,4-dihydroxybenzoic acid p-(3,4-dihydroxyphenoxy)anilide; 3,4-dihydroxybenzoic acid
o-(3,4-dihydroxyphenoxy)anilide; 2,6-bis (3,4-dihydroxyphenoxy)pyridine; 3,4-dihydroxy-benzoic
acid 3,4-dihydroxybenzylamide; 3,4-dihydroxybenzoic acid 3,4-dihydroxyphenethylamide;
3,4-dihydroxyphenyl acetic acid 3,4-dihydroxyanilide; 3,4-dihydroxyphenylacetic acid
3,4-dihydroxybenzylamide; 3,4-dihydroxyphenylacetic acid 3,4-dihydroxyphenethylamide;
3-(3,4-dihydroxyphenyl)propionic acid 3,4-dihydroxyanilide; 3-(3,4-dihydroxyphenyl)
propionic acid 3,4-dihydroxybenzylamide; 3-(3,4-dihydroxyphenyl)propionic acid 3,4-dihydroxyphenethylamide;
3,4-dihydroxycinnamic acid 3,4-dihydroxyanilide; 3,4-dihydroxycinnanic acid 3,4-dihydroxybenzylamide;
3,4-dihydroxycinnamic acid 3,4-dihydroxyphenethylamide; oxalic acid bis(3,4-dihydroxyanilide);
oxalic acid bis(3,4-dihydroxybenzyl-amide); oxalic acid bis(3,4-dihydroxyphenethylamide);
succinic acid bis(3,4-dihydroxyanilide); succinic acid bis(3,4-dihydroxybenzylamide);
succinic acid bis(3,4-dihydroxyphenethylamide); maleic acid bis(3,4-dihydroxyanilide);
maleic acid bis(3,4-dihydroxybenzylamide); fumaric acid bis(3,4-dihydroxy-anilide);
fumaric acid bis(3,4-dihydroxybenzylamide); bis(3,4-dihydroxybenzyl)amine; N-(3,4-dihydroxy-benzyl)-3,4-dihydroxyphenethylamine;
tris(3,4-dihydroxybenzyl)amine; 1,3-bis(3,4-dihydroxyphrenyl)urea; 1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxybenzyl)urea;
1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxy-phenethyl)urea; 3-deoxy-3-(3,4-dihydroxybenzyl)aminoepicatechin;
3-deoxy-3-(3,4-dihydroxyphenethyl)-aminoepicatechin; 2,3,6,7-tetrahydroxy-9,10-epoxy-9,10-dihydroacridine;
10-aminoanthracene-1,2,7,8-tetraol; acridine-1,2,6,7-tetraol; phenoxazine-2,3,7,8,10-pentaol;
dibenzo[c,f] [2,7]napthyridine-2,3,10,11-tetraol- and 6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo(de,g]quinoline-2,10,11-triol;
- (2) the methylenedioxy analogs and pharmaceutically acceptable esters thereof; and
- (3) the pharmaceutically acceptable salts of the compounds of (1) and (2).
[0045] Within this second group, a subgroup of compounds is:
- (1) the compounds that are:
3,4,3',4'-tetrahydroxybenzoin; 3,4,3',4'-tetrahydroxydesoxybenzoin; 3,4,3',4'-tetrahydroxydiphenylmethane;
1,2-bis(3,4-dihydroxyphenyl)ethane; 1,3-bis(3,4-dihydroxypheny)propane; 3,4,3',4'-tetrahydroxychalcone;
3,5-bis(3,4-dihydroxyphenyl)-1-methyl-2-pyrazoline; 4,6-bis(3,4-dihydroxyphenyl)-3-cyano-2-methylpyridine;
1,4-bis(3,4-dihydroxybenzyl)piperazine; N,N'-bis(3,4-dihydroxybenzyl)-N,N'-dimethylethylenediamine;
2,5-bis(3,4-dihydroxybenzyl)-2,5-diaza[2.2.1]bicycloheptane; N,N'-bis(3,4-dihydroxy-benzyl)-trans-1,2-diaminocyclohexane; N,N'-bis(3,4-dihydroxybenzyl)-trans-1,4-diaminocyclohexane; N,N'-bis(3,4-dihydroxybenzyl)-cis-1,3-bis(aminomethyl)cyclohexane; N-(3,4-dihydroxybenzyl)proline 3,4-dihydroxybenzylamide;
2-(3,4-dihydroxybenzyl)isoquinoline-3-carboxylic acid 3,4-dihydroxy-phenethylamide;
2,6-bis(3,4-dihydroxybenzyl)cyclohexanone; 3,5-bis(3,4-dihydroxybenzyl)-1-methyl-4-piperidinone;
2,4-bis(3,4-dihydroxybenzyl)-3-tropinone; tris(3,4-dihydroxybenzyl)methane; α-(3,4-dihydroxybenzamido)-3,4-dihydroxycinnamic
acid 3,4-dihydroxybenzyl amide; 4-(3,4-dihydroxy-benzylaminomethylene)-2-(3,4-dihydroxyphenyl)oxazolin-5-one;
1,4-bis(3,4-dihydroxybenzoyl)piperazine; N,N'-bis(3,4-dihydroxybenzoyl)-N,N'-dimethylethylenediamine;
2,5-bis(3,4-dihydroxybenzoyl)-2,5-diaza(2.2.1)bicycloheptane; N,N'-bis(3,4-dihydroxybenzoyl)-trans-1,2-diaminocyclohexane; N,N'-bis(3,4-dihydroxybenzoyl)-cis-1,3-bis(aminomethyl)cyclohexane; 3,6-bis(3,4-dihydroxybenzyl)-2,5-diketopiperazine;
3,6-bis(3,4-dihydroxybenzylidene)-1,4-dimethyl-2,5-diketopiperazine; N-(3,4-dihydroxyphenylacetyl)proline-3,4-dihydroxyanilide;
2,3-bis(3,4-dihydroxyphenyl)butane; 1,3-bis(3,4-dihydroxybenzyl)benzene;1,4-bis(3,4-dihydroxybenzyl)benzene;
2,6-bis(3,4-dihydroxybenzyl)-pyridine; 2,5-bis(3,4-dihydroxybenzyl)thiophene; 2,3-bis(3,4-dihydroxybenzyl)thiophene;
1,2-bis(3,4-dihydroxyphenyl)cyclohexane; 1,4-bis(3,4-dihydroxyphenyl)cyclohexane;
3,7-bis(3,4-dihydroxyphenyl)bicyclo[3.3.0]octane; 2,3-bis(3,4-dihydroxyphenyl)-1,7,7-trimethyl-bicyclo[2.2.1]heptane;
1,2-bis(3,4-dihpdroxyphenoxy)ethane; 1,3-bis(3,4-dihydroxyphenoxy)propane; trans-1,2-bis(3,4-dihydroxy-phenoxy)cyclopentane; N-(3,4-dihydroxybenzyl)-3-(3,4-dihydroxyphenoxy)-2-hydroxypropylamine;
3,4-dihydroxyphenoxyacetic acid 3,4-dihydroxyanilide; 3,4-dihydroxyphenoxyacetic acid
3,4-dihydroxybenzylamide; 3,4-dihydroxyphenoxyacetic acid 3,4-dihydroxyphenethylamide;
3,4-dihydroxybenzoic acid p-(3,4-dihydroxyphenoxy)anilide; 3,4-dihydroxybenzoic acid
o-(3,4-dihydroxyphenoxy)anilide; 2,6-bis(3,4-dihydroxyphenoxy)pyridine; 3,4-dihydroxybenzoic
acid 3,4-dihydroxybenzylamide; 3,4-dihydroxybenzoic acid 3,4-dihydroxyphenethylamide;
3,4-dihydroxyphenyl acetic acid 3,4-dihydroxyanilide; 3,4-dihydroxyphenylacetic acid
3,4-dihydroxybenzyl-amide; 3,4-dihydroxyphenylacetic acid 3,4-dihydroxyphenethylamide;
3-(3,4-dihydroxyphenyl)propionic acid 3,4-dihydroxyanilide; 3-(3,4-dihydroxyphenyl)
propionic acid 3,4-dihydroxybenzylamide; 3-(3,4-dihydroxyphenyl)propionic acid 3,4-dihydroxyphenethylamide;
3,4-dihydroxycinnamic acid 3,4-dihydroxyanilide; 3,4-dihydroxycinnamic acid 3,4-dihydroxybenzylamide;
3,4-dihydroxycinnamic acid 3,4-dihydroxyphenethylamide; oxalic acid bis(3,4-dihydroxyanilide);
oxalic acid bis(3,4-dihydroxybenzylamide); oxalic acid bis(3,4-dihydroxyphenethylamide);
succinic acid bis(3,4-dihydroxyanilide); succinic acid bis(3,4-dihydroxybenzylamide);
succinic acid bis(3,4-dihydroxyphenethylamide); maleic acid bis(3,4-dihydroxyanilide);
maleic acid bis(3,4-dihydroxybenzylamide); fumaric acid bis(3,4-dihydroxy-anilide);
fumaric acid bis(3,4-dihydroxybenzylamide); bis(3,4-dihydroxybenzyl)amine; N-(3,4-dihydroxybenzyl)-3,4-dihydroxyphenethylamine;
tris(3,4-dihydroxybenzyl)amine; 1,3-bis(3,4-dihydroxyphenyl)urea; 1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxybenzyl)urea;
1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxy-phenethyl)urea; 3-deoxy-3-(3,4-dihydroxybenzyl)aminoepicatechin;
and 3-deoxy-3-(3,4-dihydroxy-phenethyl)aminoepicatechin;
- (2) the methylenedioxy analogs and pharmaceutically acceptable esters thereof; and
- (3) the pharmaceutically acceptable salts of the compounds of (1) and (2).
[0046] Within this subgroup, a further subgroup is:
- (1) the compounds that are:
3,4,3',4'-tetrahydroxybenzoin; 3,4,3',4'-tetrahydroxydiphenylmethane; 1,2-bis(3,4-dihydroxyphenyl)ethane;
4,6-bis(3,4-dihydroxyphenyl)-3-cyano-2-methylpyridine, 1,4-bis(3,4-dihydroxybenzyl)piperazine;
N,N'-bis(3,4-dihydroxybenzyl)-trans-1,2-diaminocyclohexane; 2,4-bis(3,4-dihydroxybenzyl)-3-tropinone; α-(3,4-dihydroxybenzamido)-3,4-dihydroxycinnamic
acid 3,4-dihydroxybenzyl amide; 1,4-bis(3,4-dihydroxybenzoyl)piperazine; N,N'-bis(3,4-dihydroxybenzoyl)-trans-1,2-diaminocyclohexane; 3,4-dihydroxybenzoic acid 3,4-dihydroxybenzylamide; 3-(3,4-dihydroxyphenyl)propionic
acid 3,4-dihydroxyanilide; 3-(3,4-dihydroxyphenyl)propionic acid 3,4-dihydroxybenzylamide;
3,4-dihydroxycinnamic acid 3,4-dihydroxybenzylamide; oxalic acid bis(3,4-dihydroxyanilide);
succinic acid bis(3,4-dihydroxyanilide); succinic acid bis(3,4-dihydroxybenzylamide),
bis(3,4-dihydroxybenzyl)amine; tris(3,4-dihydroxybenzyl)amine; 1,3-bis(3,4-dihydroxyphenyl)urea;
and 1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenethyl)urea;
- (2) the methylenedioxy analogs and pharmaceutically acceptable esters thereof; and
- (3) the pharmaceutically acceptable salts of the compounds of (1) and (2).
Synthesis of the compounds
[0047] The compound of this invention and the compounds disclosed above may be prepared
by methods generally known to the person of ordinary skill in the art, having regard
to that knowledge and the disclosure of this application including Examples 1-24.
[0048] The starting materials and reagents used in preparing these compounds are either
available from commercial suppliers such as the Aldrich Chemical Company (Milwaukee,
WI), Bachem (Torrance, CA), Sigma (St Louis, MO), or Lancaster Synthesis Inc. (Windham,
NH) or are prepared by methods well known to a person of ordinary skill in the art,
following procedures described in such references as
Fieser and Fieser's Reagents for Organic Synthesis, vols. 1-17, John Wiley and Sons,
New York, NY, 1991;
Rodd's Chemistry of Carbon Compounds, vols. 1-5 and supps., Elsevier Science Publishers,
1989;
Organic Reactions, vols.1-40, John Wiley and Sons, New York, NY, 1991;
March J.: Advanced Organic Chemistry, 4th ed., John Wiley and Sons, New York, NY; and
Larock: Comprehensive Organic Transformations, VCH Publishers, New York, 1989.
[0049] In most cases, protective groups for the hydroxy groups are introduced and finally
removed. Suitable protective groups are described in
Greene et al., Protective Groups in Organic Synthesis, Second Edition, John Wiley
and Sons, New York, 1991. A preferred protective group is the methylenedioxy group, as seen in many of Examples
1-23, and a wide variety of methylenedioxyphenyl compounds (such as 3,4-methylenedioxyacetophenone,
3,4-methylenedioxyaniline, 3,4-methylenedioxybenzaldehyde, 3,4-methylenedioxybenzoic
acid, 3,4-methylenedioxybenzonitrile, 3,4-methylenedioxybenzoic acid, 3,4-methylenedioxybenzoyl
chloride, 3,4-methylenedioxycinnamic acid, 3,4-methylenedioxynitrobenzene, 3,4-methylenedioxyphenol,
3,4-methylenedioxyphenylacetic acid, 3,4-methylenedioxyphenylacetonitrile, 3,4-methylenedioxyphenyl
isocyanate, 3,4-methylenedioxyphenylmagnesium bromide, and 3,4-methylenedioxyphenylmethanol)
are commercially available. Other protecting groups, such as the benzyl and methoxymethyl
groups, may also be used.
[0050] O ther starting materials or early intermediates may be prepared by elaboration of
the materials listed above, for example, by methods well known to a person of ordinary
skill in the art
[0051] The starting materials, intermediates, and compounds may be isolated and purified
using conventional techniques, including precipitation, filtration, distillation,
crystallization, chromatography, and the like. The compounds may be characterized
using conventional methods, including physical constants and spectroscopic methods.
Pharmacology and Utility
[0052] The compounds and pharmaceutically acceptable salts thereof disclosed herein act
to inhibit or prevent amyloid fibril formation, inhibit or prevent amyloid fibril
growth, and/or cause disassembly, disruption, and/or disaggregation of pre-formed
amyloid fibrils and amyloid protein deposits. Their activity can be measured
in vitro by methods such as those discussed in Examples 25-27, while their activity
in vivo against amyloid diseases can be measured in animal models, such as those APP transgenic
mouse models that mimic many of the neuropathological hallmarks of Alzheimer's disease,
and in humans.
[0053] "Amyloid diseases" or "amyloidosis" suitable for treatment with the compounds disclosed
herein ate diseases associated with the formation, deposition, accumulation, or persistence
of amyloid fibrils, especially the fibrils of an amyloid protein selected from the
group consisting of Aβ amyloid, AA amyloid, AL amyloid, IAPP amyloid, PrP amyloid,
α
2-microglobulin amyloid, transthyretin, prealbumin, and procalcitonin, especially Aβ
amyloid and IAPP amyloid. Suitable such diseases include Alzheimer's disease,' Down's
syndrome, dementia pugilistica, multiple system atrophy, inclusion body myositosis,
hereditary cerebral hemorrhage with amyloidosis of the Dutch type, Nieman-Pick disease
type C, cerebral β-amyloid angiopathy, dementia associated with cortical basal degeneration,
the amyloidosis of type 2 diabetes, the amyloidosis of chronic inflammation, the amyloidosis
of malignancy and Familial Mediterranean Fever, the amyloidosis of multiple myeloma
and B-cell dyscrasias, the amyloidosis of the prion diseases, Creutzfeldt-Jakob disease,
Gerstmann-Straussler syndrome, kuru, scrapie, the amyloidosis associated with carpal
tunnel syndrome, senile cardiac amyloidosis, familial amyloidotic polyneuropathy,
and the amyloidosis associated with endocrine tumors, especially Alzheimer's disease
and type 2 diabetes.
[0054] The compounds also act to inhibit or prevent α-synuclein/NAC fibril formation, inhibit
or prevent α-synuclein/NAC fibril growth, and/or cause disassembly, disruption, and/or
disaggregation of preformed α-synuclein/NAC fibrils and α-synuclein/NAC-associated
protein deposits. Their activity can be measured
in vitro by methods similar to those discussed in Examples 24-26, or
in vivo in animal models, such as those α-synuclein transgenic mouse models that mimic some
of the neuropathological hallmarks of Parkinson's disease, and in humans.
[0055] "Synuclein diseases" or "synucleinopathies" suitable for treatment with the compounds
of this invention are diseases associated with the formation, deposition, accumulation,
or persistence of synuclein fibrils, especially α-synuclein fibrils. Suitable such
diseases include Parkinson's disease, familial Parkinson's disease, Lewy body disease,
the Lewy body variant of Alzheimer's disease, dementia with Lewy bodies, multiple
system atrophy, and the Parkinsonism-dementia complex of Guam.
[0056] The therapeutic ratio of a compound can be determined, for example, by comparing
the dose that gives effective anti-fibril (anti-amyloid or anti-α-synuclein/NAC) activity
in a suitable
in vivo model in a suitable animal species such as the mouse, with the dose that gives significant
weight loss (or other observable side-effects) in the test animal species.
[0057] Compounds and pharmaceutically acceptable salts thereof which are useful in treating
an amyloid disease or a synucleinopathy in a mammal suffering therefrom, or which
are useful in treating the formation, deposition, accumulation or persistence of amyloid
fibrils or synuclein fibrils are selected from the group consisting of compounds of
the formula:
where:
R is a C2-C10 alkylene group, in which there is optionally 1 double bonds; 1 or 2 non-adjacent
ethylene groups are replaced by-C(O)NR'- or -NR'C(O)-, where R' is H or a lower alkyl.
[0058] In certain embodiments, the amyloid disease is a disease associated with the formation,
deposition, accumulation, or persistence of an amyloid protein selected from the group
consisting of Aβ amyloid, AA amyloid, AL amyloid, IAPP amyloid, PrP amyloid, α
2-microglobulin amyloid, transthyretin, prealbumin, and procalcitonin.
[0059] The amyloid disease may be selected from the group of :
diseases consisting of Alzheimer's disease, Down's syndrome, dementia pugilistica,
multiple system atrophy, inclusion body myositosis, hereditary cerebral hemorrhage
with amyloidosis of the Dutch type, Nieman-Pick disease type C, cerebral β-amyloid
angiopathy, dementia associated with cortical basal degeneration, the amyloidosis
of type 2 diabetes, the amyloidosis of chronic inflammation, the amyloidosis of malignancy
and Familial Mediterranean Fever, the amyloidosis of multiple myeloma and B-cell dyscrasias,
the amyloidosis of the prion diseases, Creutzfeldt-Jakob disease, Gerstmann-Straussler
syndrome, kuru, scrapie, the amyloidosis associated with carpal tunnel syndrome, senile
cardiac amyloidosis, familial amyloidotic polyneuropathy, and the amyloidosis associated
with endocrine tumors.
[0060] In certain embodiments, the synucleinopathy is a disease associated with the formation,
deposition, accumulation, or persistence of synuclein fibrils, preferably α-synuclein
fibrils.
[0061] The synucleinopathy may be selected from the group of diseases consisting of Parkinson's
disease, familial Parkinson's disease, Lewy body disease, the Lewy body variant of
Alzheimer's disease, dementia with Lewy bodies, multiple system atrophy, and the Parkinsonism-dementia
complex of Guam.
[0062] When the disease to be treated is a synucleinopathy associated with the formation,
deposition, accumulation or persistence of synuclein fibrils the compound is preferably
selected from the group consisting of: 3,4-dihydroxybenzoic acid 3,4-dihydroxyanilide;
3,4-dihydroxybenzoic acid 3,4-dihydroxybenzylamide; succinic acid bis(3,4-dihydroxyanilide);
and pharmaceutically acceptable salts thereof.
[0063] preferably the mammal to be treated by the present invention is a human.
[0064] Preferably, the amyloid fibrils are Aβ amyoid fibris, or IAPP amyloid fibrils.
[0065] preferably, the synuclein fibrils are α-synuclein fibrils.
[0066] Compounds of special interest for treating the formation, deposition, accumulation,
or persistence of Aβ amyloid fibrils, or for treating Alzheimer's disease, are selected
from the group consisting of
- (1) the compounds that are:
3,4,3',4'-tetrahydroxybenzoin; 3,4,3',4'-tetrahydroxydiphenylmethane;1,2-bis(3,4-dihydroxyphenyl)ethane;
N,N'-bis(3,4-dihydroxybenzyl)-trans-1,2-diaminocyclohexane; α-(3,4-dihydroxybenzamido)-3,4-dihydroxycinnamic acid 3,4-dihydroxybenzylamide;
3,4-dihydroxybenzoic acid 3,4-dihydroxyanilide; bis(3,4-dihydroxybenzyl)amine; 1,3-bis(3,4-dihydroxyphenyl)urea;
and 1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenethyl)urea;
- (2) the pharmaceutically acceptable salts of the compounds of (1)
[0067] Compounds of special interest for treating the formation, deposition, accumulation,
or persistence of IAPP amyloid fibrils, or for treating type 2 diabetes, are selected
from the group consisting of
- (1) the compounds that are:
3,4,3',4'-tetrahydroxybenzoin; 3,4,3',4'-tetrahydroxydiphenylmethane; 1,2-bis(3,4-dihydroxyphenyl)ethane;
2,4-bis(3,4-dihydroxybenzyl)-3-tropinone;1,4-bis(3,4-dihydroxybenzoyl)piperazine;
3,4-dihydroxybenzoic acid 3,4-dihydroxyanilide; 3,4-dihydroxybenzoic add 3,4-dihydroxybenzylamide;
3-(3,4-dihydroxyphenyl)propionic acid 3,4-dihydroxybenzylamide; oxalic add bis(3,4-dihydroxyanilide);
succinic acid bis(3,4-dihydroxyanilide); tris(3,4-dihydroxybenzyl)amine; and 1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenethyl)urea;
- (2) the pharmaceutically acceptable salts of the compounds of (1)
[0068] Compounds of special interest for treating the formation, deposition, accumulation,
or persistence of α-synuclein fibrils, or for treating Parkinson's disease or other
synucleinopathies, are selected from the group consisting of
- (1) the compounds that are:
3,4,3',4'-tetrahydroxybenzoin; 3,4,3',4'-tetrahydroxydiphenylmethane;1,2-bis(3,4-dihydroxyphenyl)ethane;
N,N'-bis(3,4-dihydroxybenzyl)-trans-1,2-diaminocyclohexane; N,N'-bis(3,4-dihydroxybenzoyl)-trans-1,2-diaminocyclohexane; 3,4-dihydroxybenzoic acid 3,4-dihydroxyanilide; 3,4-dihydroxybenzoic
acid 3,4-dihydroxybenzylamide; succinic acid bis(3,4-dihydroxyanilide); bis(3,4-dihydroxybenzyl)amine;
and 1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenethyl)urea;
- (2) the pharmaceutically acceptable salts of the compounds of (1)
Pharmaceutical Compositions and Administration
[0069] In general, compounds will be administered in therapeutically effective amounts by
any of the usual modes known in the art, either singly or in combination with at least
one other compound of this invention and/or at least one other conventional therapeutic
agent for the disease being treated. A therapeutically effective amount may vary widely
depending on the disease, its severity, the age and relative health of the animal
being treated, the potency of the compound(s), and other factors. As anti-fibril agents,
therapeutically effective amounts of the compounds may range from 0.1-1000 mg/Kg body
weight/day, such as from 1-100 mg/Kg/day; for example, 10-100 mg/Kg/day. A person
of ordinary skill in the art will be conventionally able, and without undue experimentation,
having regard to that skill and to this disclosure, to determined a therapeutically
effective amount of a compound for the treatment of an amyloid disease such as an
amyloidosis or α-synuclein/NAC fibril formation.
[0070] Preferred compositions will contain a compound that is at least substantially pure.
In general "pure" means better than 95% pure, and "substantially pure" means a compound
synthesized such that the compound, as made as available for consideration into a
therapeutic dosage, has only those impurities that can not readily nor reasonably
be removed by conventional purification processes.
[0071] In general, the compounds will be administered as pharmaceutical compositions by
one of the following routes: oral, topical, systemic (e.g. transdermal, intranasal,
or by suppository), or parenteral (e.g. intramuscular, subcutaneous, or intravenous
injection). Compositions may take the form of tablets, pills, capsules, semisolids,
powders, sustained release formulations, solutions, suspensions, elixirs, aerosols,
or any other appropriate compositions; and comprise at least one compound in combination
with at least one pharmaceutically acceptable excipient. Suitable excipients are well
known to persons of ordinary skill in the art, and they, and the methods of formulating
the compositions, may be found in such standard references as
Remington: The Science and Practice of Pharmacy, A. Gennaro, ed., 20th edition, Lippincott,
William & Wilkins, Philadelphia, PA. Suitable liquid carriers, especially for injectable solutions, include water, aqueous
saline solution, aqueous dextrose solution, and glycols.
[0072] In particular, the compound(s) - optimally only one such compound is administered
in any particular dosage form - can be administered; orally, for example, as tablets,
troches, lozenges, aqueous or oily suspension, dispersible powders or granules, emulsions,
hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may
be prepared according to any method known in the art for the manufacture of pharmaceutical
compositions and such compositions may contain one or more agents selected from the
group consisting of sweetening agents, flavoring agents, coloring agents and preserving
agents in order to provide pharmaceutically elegant and palatable preparations.
[0073] Tablets contain the compound in admixture with non-toxic pharmaceutically acceptable
excipients that are suitable for the manufacture of tablets. These excipients may
be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose,
calcium phosphate or sodium phosphate; granulating and disintegrating agents, for
example, maize starch or alginic acid; binding agents, for example, maize starch,
gelatin or acacia, and lubricating agents, for example, magnesium stearate or stearic
acid or tale. The tablets may be uncoated or they may be coated by known techniques
to delay disintegration and absorption in the gastrointestinal tract and thereby provide
a sustained action over a longer period. For example, a time delay material such as
glycerol monostearate or glycerol distearate may be employed. Formulations for oral
use may also be presented as hard gelatin capsules wherein the compound is mixed with
an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin,
or as soft gelatin capsules wherein the active ingredient is mixed with water or an
oil medium, for example, peanut oil, liquid paraffin or olive oil.
[0074] Aqueous suspensions contain the compound in admixture with excipients suitable for
the manufacture of aqueous suspensions. Such excipients are suspending agents, for
example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl cellulose,
sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia; dispersing
or wetting agents may be naturally occurring phosphatides, for example lecithin, or
condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene
stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols,
for example, heptadecaethyleneoxycetanol, or condensation products of ethylene oxide
with partial esters derived from fatty acids such as hexitol such as polyoxyethylene
sorbitol monooleate, or condensation products of ethylene oxide with partial esters
from fatty acids and a hexitol anhydrides, for example, polyethylene sorbitan monooleate.
The aqueous suspensions may also contain one or more preservatives, for example, ethyl
or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring
agents, or one or more sweetening agents, such as sucrose or saccharin.
[0075] Oily suspensions may be formulated by suspending the compound in a vegetable oil,
for example arachis oil, olive oil, sesame oil, or coconut oil or in a mineral oil
such as liquid paraffin. The oily suspensions may contain a thickening agent, for
example beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those
set forth below, and flavoring agents may be added to provide a palatable oral preparation.
These compositions may be preserved by the addition of an antioxidant such as ascorbic
acid. Dispersible powders and granules suitable for preparation of an aqueous suspension
by the addition of water provide the active ingredient in admixture with a dispersing
or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing
or wetting agents and suspending agents are exemplified by those already described
above. Additional excipients, for example sweetening, flavoring and agents, may also
be present
[0076] The compounds may also be in the form of oil-in-water emulsions. The oily phase may
be a vegetable oil, for example olive oil or arachis oils, or a mineral oil, for example
liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring
gums, for example gum acacia or gum tragacanth, naturally occurring phosphatides,
for example soy bean, lecithin, and occurring phosphatides, for example soy bean,
lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides,
for example sorbitan monooleate, and condensation products of the said partial esters
with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsion
may also contain sweetening and flavoring agents. Syrups and elixirs may be formulated
with sweetening agents, for example, glycerol, sorbitol or sucrose. Such formulations
may also contain a demulcent, a preservative and flavoring and coloring agents.
[0077] The compounds can also be administered by injection or infusion, either subcutaneously
or intravenously, or intramuscularly, or intrasternally, or intranasally, or by infusion
techniques in the form of sterile injectable or oleaginous suspension. The compound
may be in the form of sterile injectable aqueous or oleaginous suspensions. These
suspensions may be formulated according to the known art using suitable dispersing
of wetting agents and suspending agents that have been described above. The sterile
injectable preparation may also be a sterile injectable solution or suspension in
a non-toxic parenterally-acceptable diluent or solvent for example, as a solution
in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed
are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile,
fixed oils are conventionally employed as a solvent or suspending medium. For this
purpose any bland fixed oils may be conventionally employed including synthetic mono-
or diglycerides. In addition fatty acids such as oleic acid find use in the preparation
of injectables. Dosage regimens can be adjusted to provide the optimum therapeutic
response. For example, several divided dosages may be administered daily or the dosage
may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
[0078] It is especially advantageous to formulate the compounds in dosage unit form for
ease of administration and uniformity of dosage. Dosage unit form as used herein refers
to physically discrete units suited as unitary dosages for the subjects to be treated;
each containing a therapeutically effective quantity of the compound and at least
one pharmaceutical excipient A drug product will comprise a dosage unit form within
a container that is labeled or accompanied by a label indicating the intended method
of treatment, such as the treatment of an amyloid disease, for example an amyloidosis
such as Alzheimer's disease or a disease associated with α-synuclein/NAC fibril formation
such as Parkinson's disease.
Sustained Release Formulations
[0079] Sustained release formulations can be used to deliver the compounds to the desired
target (i.e. brain or systemic organs) at high circulating levels (between 10
-9 and 10
-4 M) are also disclosed. In a preferred embodiment for the treatment of Alzheimer's
or Parkinson's disease, the circulating levels of the compounds is maintained up to
10
-7 M. The levels are either circulating in the patient systemically, or in a preferred
embodiment, present in brain tissue, and in a most preferred embodiments, localized
to the amyloid or α-synuclein fibril deposits in brain or other tissues.
[0080] It is understood that the compound levels are maintained over a certain period of
time as is desired and can be easily determined by one skilled in the art using this
disclosure and compounds of the invention. A unique feature of this administration
is disclosed comprising a sustained release formulation so that a constant level of
therapeutic compound is maintained between 10
-8 and 10
-6 M between 48 to 96 hours in the sera.
[0081] Such sustained and/or timed release formulations may be made by sustained release
means of delivery devices that are well known to those of ordinary skill in the art,
such as those described in
US Patent Nos. 3,845,770;
3,916,899;
3,536,809;
3, 598,123;
4,008,719;
4,710,384;
5,674,533;
5,059,595;
5,591,767;
5,120,548;
5,073,543;
5,639,476;
5,354,556 and
5,733,566.
[0082] These pharmaceutical compositions can be used to provide slow or sustained release
of one or more of the active compounds using, for example, hydroxypropylmethyl cellulose,
other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings,
microparticles, liposomes, microspheres, or the like. Suitable sustained release formulations
known to those skilled in the art, including those described herein, may be readily
selected for use with the pharmaceutical compositions of the invention. Thus, single
unit dosage forms suitable for oral administration, such as, but not limited to, tablets,
capsules, gelcaps, caplets, powders and the like, that are adapted for sustained release
are encompassed by the present invention.
[0083] The sustained release formulation may contain active compounds such as, but not limited
to, microcrystalline cellulose, maltodextrin, ethylcellulose, and magnesium stearate.
As described above, all known methods for encapsulation which are compatible with
properties of the disclosed compounds are encompassed by this disclosure. The sustained
release formulation is encapsulated by coating particles or granules of the pharmaceutical
composition of the invention with varying thickness of slowly soluble polymers or
by microencapsulation. The sustained release formulation may be encapsulated with
a coating material of varying thickness (e.g. about 1 micron to 200 microns) that
allow the dissolution of the pharmaceutical composition about 48 hours to about 72
hours after administration to a mammal. The coating material may be a food-approved
additive.
[0084] The sustained release formulation may be a matrix dissolution device that is prepared
by compressing the drug with a slowly soluble polymer carrier into a tablet
[0086] Each of the particles is in the form of a micromatrix, with the active ingredient
uniformly distributed throughout the polymer.
[0087] Sustained release formulations such as those described in
U.S. Patent No. 4,710,384, having a relatively high percentage of plasticizer in the coating in order to permit
sufficient flexibility to prevent substantial breakage during compression are disclosed.
The specific amount of plasticizer varies depending on the nature of the coating and
the particular plasticizer used. The amount may be readily determined empirically
by testing the release characteristics of the tablets formed. If the medicament is
released too quickly, then more plasticizer is used. Release characteristics are also
a function of the thickness of the coating. When substantial amounts of plasticizer
are used, the sustained release capacity of the coating diminishes. Thus, the thickness
of the coating may be increased slightly to make up for an increase in the amount
of plasticizer. Generally, the plasticizer in such an embodiment will be present in
an amount of about 15 to 30 %of the sus tained release material in the coating, preferably
20 to 25 %, and the amount of coating will be from 10 to 25% of the weight of the
active material, preferably 15 to 20 %. Any conventional pharmaceutically acceptable
plasticizer may be incorporated into the coating.
[0088] The compounds can be formulated as a sustained and/or timed release formulation.
All sustained release pharmaceutical products have a common goal of improving drug
therapy over that achieved by their non-sustained counterparts. Ideally, the use of
an optimally designed sustained release preparation in medical treatment is characterized
by a minimum of drug substance being employed to cure or control the condition. Advantages
of sustained release formulations may include: 1) expended activity of the composition,
2) reduced dosage frequency, and 3) increased patient compliance. In addition, sustained
release formulations can be used to affect the time of onset of action or other characteristics,
such as blood levels of the composition, and thus can affect the occurrence of side
effects.
[0089] The sustained release formulations are designed to initially release an amount of
the therapeutic composition that promptly produces the desired therapeutic effect,
and gradually and continually release of other amounts of compositions to maintain
this level of therapeutic effect over an extended period of time. In order to maintain
this constant level in the body, the therapeutic composition must be released from
the dosage form at a rate that will replace the composition being metabolized and
excreted from the body.
[0090] The sustained release of an active ingredient may be stimulated by various inducers,
for example pH, temperature, enzymes, water, or other physiological conditions or
compounds. The term "sustained release component" in the context of the present disclosure
defined herein as a compound or compounds, including, but not limited to, polymers,
polymer matrices, gels, permeable membranes, liposomes, microspheres, or the like,
or a combination thereof, that facilitates the sustained release of the active ingredient.
[0091] If the complex is water-soluble, it may be formulated in an appropriate buffer, for
example, phosphate buffered saline, or other physiologically compatible solutions.
Alternatively, if the resulting complex has poor solubility in aqueous solvents, then
it may be formulated with a non-ionic surfactant such as Tween, or polyethylene glycoL
Thus, the compounds and their physiologically solvents may be formulated for administration
by inhalation or insufflation (either through the mouth or the nose) or oral, buccal,
parenteral, or rectal administration, as examples.
[0092] Preparations for oral administration may be suitably formulated to give controlled
release of the active compound. In a preferred embodiment, the compounds are formulated
as controlled release powders of discrete microparticles that can be readily formulated
in liquid form. The sustained release powder comprises particles containing an active
ingredient and optionally, an excipient with at least one non-toxic polymer.
[0093] The powder can be dispersed or suspended in a liquid vehicle and will maintain its
sustained release characteristics for a useful period of time. These dispersions or
suspensions have both chemical stability and stability in terms of dissolution rate.
The powder may contain an excipient comprising a polymer, which may be soluble, insoluble,
permeable, impermeable, or biodegradable. The polymers may be polymers or copolymers.
The polymer may be a natural or synthetic polymer. Natural polymers include polypeptides
(e.g., zein), polysaccharides (e.g., cellulose), and alginic acid. Representative
synthetic polymers include those described, but not limited to, those described in
column 3, lines 33-45 of
U.S. Patent No. 5,354,556, Particularly suitable polymers include those described, but not limited to those
described in column 3, line 46-column 4, line 8 of
U.S. Patent No. 5,354,556
[0094] The sustained release compounds of the invention may be formulated for parenteral
administration, e.g., by intramuscular injections or implants for subcutaneous tissues
and various body cavities and transdermal devices. In one embodiment, intramuscular
injections are formulated as aqueous or oil suspensions. In an aqueous suspension,
the sustained release effect is due to, in part, a reduction in solubility of the
active compound upon complexation or a decrease in dissolution rate. A similar approach
is taken with oil suspensions and solutions, wherein the release rate of an active
compound is determined by partitioning of the active compound out of the oil into
the surrounding aqueous medium. Only active compounds which are oil soluble and have
the desired partition characteristics are suitable. Oils that may be used for intramuscular
injection include, but are not limited to, sesame, olive, arachis, maize, almond,
soybean, cottonseed and castor oil.
[0095] A highly developed form of drug delivery that imparts sustained release over periods
of time ranging from days to years is to implant a drug-bearing polymeric device subcutaneously
or in various body cavities. The polymer material used in an implant, which must be
biocompatible and nontoxic, include but are not limited to hydrogels, silicones, polyethylenes,
ethylene-vinyl acetate copolymers, or biodegradable polymers.
EXAMPLES
General Experimental Procedures
[0096] All solvents were distilled before use and were removed by rotary evaporation at
temperatures up to 35°C. Octadecyl functionalized silica gel (C18) was used for reversed-phase
(RP) flash chromatography, and Merck silica gel 60, 200-400 mesh, 40-63 µm, was used
for silica.gel flash chromatography. Thin layer chromatography (TLC) was carried out
using Merck DC-plastikfolien Kieselgel 60 F
254, first visualized with a UV lamp, and then by dipping in a vanillin solution (1%
vanillin, 1% H
2SO
4 in ethanol), and heating. Optical rotations were measured on a Perkin-Elmer 241 polarimeter.
Mass spectra were recorded on a Kratos MS-80 instrument NMR spectra, at 25°C, were
recorded at 500 or 300 MHz for
1H and 125 or 75 MHz for
13C on Varian INOVA-500 or VXR-300 spectrometers. Chemical shifts are given in ppm on
the delta scale referenced to the solvent peaks CHCl
3 at 7.25 and CDCl
3 at 77.0 ppm, (CH
3)
2CO at 2.15 and (CD
3)
2CO at 30.5 ppm, or CH
3OD at 3.30 and CD
3OD at 39.0 ppm.
HPLC Conditions
[0097] The analytical HPLC equipment consisted of a Waters 717 autosampler, 600 pump and
controller, and a 2487 UV detector controlled by Omega software. Samples were analyzed
by using an RP-18 semi-preparative column (Phenomenex Prodigy 5 mm C18 100A, 250 x
4.6 mm) with a guard column (Phenomenex SecurityGuard cartridge containing a C18 ODS
4 x 3 mm, 5 mm column) fitted at 30°C. Samples (5 ml) were analyzed using a mobile
phase flow rate of 5.0 ml/min, with UV detection at 280 nm.
Method 1
[0098]
Time (minutes) |
CH3CN |
H2O containing 0.1% TFA |
0 |
11 |
89 |
20 |
11 |
89 |
30 |
100 |
0 |
31 |
11 |
89 |
40 |
11 |
89 |
Method 2
[0099]
Time (minutes) |
CH3CN/H2O (95:5) containing 0.1% TFA |
H2O containing 0.1% TFA |
0 |
11 |
89 |
20 |
11 |
89 |
30 |
100 |
0 |
31 |
11 |
89 |
40 |
11 |
89 |
Comparative Example 1: 3,4,3',4'-Tetrahydroxybenzoin (Compound 1; DC-0001) Bis(3,4-methylenedioxy)benzoin
(compound 1B; DC-0001B)
[0100]
[0101] A solution of piperonal (5 g) in ethanol (6.5 ml) was treated with a solution of
potassium cyanide (0.5 g) in water (5 ml), then refluxed for 5 h. The resultant precipitate
was filtered off, washed with water then crystallized from ethanol to give
DC-0001B (2.24 g, 45%) as an off white crystalline solid.
1H-NMR(CDCl
3) 7.52 (1H, dd, J 2, 8Hz), 7.39 (1H, d, J 2Hz), 6.73 - 6.82 (4H, m), 6.02 (2H, s),
5.91 (2H, m), 5.76 (1H, d, J 6Hz) and 4.51 (1H, d, J 6Hz).
M/z 287 ((M - CH)-, 100%).
Bis(3,4-methylenedioxy)benzil
[0102] A mixture of copper acetate (20 mg), ammonium nitrate (660 mg) and
DC-0001B (2 g) in aq. acetic acid (80%, 10 ml) were refluxed together for 90 minutes. The
mixture was cooled then poured into water (100 ml) and the product extracted into
ethyl acetate (2 x 100 ml), dried and evaporated in vacuo to give a yellow gum. Trituration
from ethanol gave bis(3,4-methylenedioxy)benzil (1.35 g, 68%) as a pale yellow solid.
1H-NMR 7.48 (2H, dd, J 2, 8Hz) 7.47 (2H, d, J 2Hz), 6.86 (2H, d, J 8Hz) and 6.08 (4H,
s).
3,4,3',4'-Tetrahydroxybenzil
[0103] To a stirred solution of bis(3,4-methylenedioxy)benzil (500 mg) in dry CH
2Cl
2 (50 ml) under nitrogen, was slowly added boron tribromide (1.6 ml) then stirring
continued for a further 3.5 hours. Methanol (100 ml) was added carefully, then the
solvent evaporated in vacuo to a volume of 1 ml, this addition and evaporation was
repeated twice more. The product was purified by column chromatography over silica
gel when elution with diethylether in dichloromethane gave 3,4,3',4'-tetrahydroxybenzil
(217 mg, 47%) as a yellow powder.
1H-NMR 9.35 (2H, bs), 8.80 (2H, bs), 7.48 (2H, d, J 2Hz), 7.34 (2H, dd, J 2, 8Hz) and
7.02 (2H, d, J 8Hz). M/z 273 ((M-H)
+, 100%).
HPLC (method 2) 31.3 minutes.
3,4,3',4'-Tetrahydroxybenzoin (Compound 1; DC-0001)
[0104] A solution of the tetrahydroxybenzil (200 mg) in methanol (20 ml) with palladium
hydroxide on carbon (20%, 10 mg) was stirred under an atmosphere of hydrogen for 5
minutes. The mixture was filtered through Celite, and the solvents removed in vacuo
to give an orange gum. Separation by column chromatography over silica gel eluting
with 20% ethyl acetate in dichloromethane gave DC-0001 as an off-white gum (55 mg,
27%). Recrystallization from methanol/dichloromethane gave pure DC-0001 as an off-white
powder (27 mg, 13%).
1H-NMR ((CD
3)
2CO) 7.41 (1H, d, J 2Hz), 7.35 (1H, dd, J 2, 8Hz), 6.75 (1H, d, J 8Hz), 6.73 (1H, d,
J 2Hz), 6.69 (1H, d, J 8Hz), 6.64 (1H, dd, J 2, 8Hz), 5.69 (1H, bd) and 4.60 (1H,
bd).
13C-NMR ((CD
3)
2CO) 198.22, 151.41, 145.77, 145.68, 145.43, 132.79, 127.07, 123.92, 120.52, 116.69,
116.20, 115.59, 115.36 and 75.97. M/Z 275 ((M-H)
+, 100%).
HPLC (Method 1) 7.1 minutes.
Comparative Example 2: 3,4,3',4'-Tetrahydroxydiphenylmethane (compound 3; DC-0003)
[0105]
Bis(3,4-methylenedioxyphenyl)methanol
[0106] To a solution of piperonal (0.75g) in solution in dichloromethane (25 ml) was added
dropwise 3,4-(methylenedioxy)phenylmagnesium bromide (5 ml, 1M solution in toluene/THF).
The mixture was stirred at room temperature overnight, then poured onto water, extracted
with dichloromethane, dried and evaporated in vacuo to give the crude alcohol as a
brown gum. Purification by column chromatography over silica gel eluting with ethyl
acetate in CH
2Cl
2 (10 to 20%) gave the pure alcohol as a white gum (1.18 g, 87%).
1H-NMR (CDCl
3) 6.7-6.8 (6H, m), 5.93 (4H, s), 5.66 (1H, bs) and 2.18 (bs).
Bis(3,4-methylenedioxyphenyl)methane (compound 3B; DC-0003B)
[0107] A solution of the alcohol (2.61 g) in methanol (25 ml)/tetrahydrofuran (30 ml) was
shaken with Pd(OH)
2/C (20%, 100 mg) under an atmosphere of hydrogen for 12 hours. The mixture was filtered
through Celite, then the solvents removed in vacuo to give a brown gum (2.4 g). Crystallization
from acetone gave
DC-0003B as white crystals (1.14 g, 44%).
1H-NMR (CDCl
3) 6.6-6.8 (6H, m), 5.90 (4H, s) and 3.79(2H, s).
3,4,3',4'-Tetrahydroxydiphenylmethane (compound 3; DC-0003)
[0108] To a stirred solution of
DC-0003B (0.214 mg) in dry CH
2Cl
2 (25 ml) under nitrogen, was slowly added boron tribromide (0.4 ml) then stirring
was continued for a further 3.5 hours. Methanol (50 ml) was added carefully, then
the solvent evaporated in vacuo to a volume of 1 ml; this was then repeated 2 more
times. The product was purified by column chromatography over silica gel when elution
with ethyl acetate in dichloromethane gave
DC-0003 (48%) as an off-white solid.
1H-NMR ((CD
3)
2CO) 7.73 (2H, s), 7.66 (2H, s), 6.74 (2H, d, 8Hz), 6.67 (2H, d, J 2Hz), 6.56 (2H,
dd, J 2, 8Hz) and 3.70 (2H, s).
13C-NMR ((CD
3)
2CO) 146.51, 144.80, 135.34, 121.59, 117.45, 116.64 and 41.90. M/z 232 (M
+, 100%).
HPLC (Method 1) 31.1 minutes.
Comparative Example 3: 1,2-bis(3,4-dihydroxyphenyl)ethane (compound 4; DC-0004)
[0109]
1,2-bis-(3,4-dihydroxyphenyl)ethane (compound 4; DC-0004)
[0110] A solution of the tetrahydroxybenzil (see Example 1) (70 mg) in methanol (10 ml)
with palladium hydroxide on carbon (20%, 10 mg) was stirred under an atmosphere of
hydrogen for 2 hours. The mixture was filtered through Celite, and the solvents removed
in vacuo to give an orange gum. Separation by column chromatography over silica gel
eluting with 20% ethyl acetate in dichloromethane gave
DC-0004 as an off white gum (43 g, 68%).
1H-NMR ((CD
3)
2CO) 7.73 (4H, bs), 6.80 (2H, d, J 8Hz), 6.79 (2H, d, J 2Hz), 6.62 (2H, dd, J 2, 8Hz)
and 2.79 (4H, s). M/z 245 ((M-H)
+, 100%).
HPLC (Method 2) 31.7 minutes.
Comparative Example 4: 4,6-bis(3,4-dihydroxyphenyl)-3-cyano-2-methylpyridine (compound 8; DC-0008)
[0111]
4,6-bis(3,4-methylenedioxyphenyl)-3-cyano-2-methylpyridine (compound 8B; DC-0008B)
[0112] To a solution of the chalcone (
see below) (300 mg, 1.0 mmol) and 3-aminocrotonitrile (82 mg, 1.2 mmol) in dry acetonitrile
was added potassium tert-butoxide (560 mg) and the mixture stirred for 18 h. The mixture
was then poured into water, extracted with ethyl acetate, dried and evaporated in
vacuo. Recrystallization from dichloromethane/ether gave
DC-0008B (152 mg, 42%) as an off-white powder.
1H-NMR(CDCl
3) 7.60 (2H, m), 7.52 (1H, s), 7.10 (2H, m), 6.93 (2H, m), 6.07 (2H, s), 6.05 (2H,
s) and 2.87 (3H, s).
M/z 359 ((M+1)
+, 100%).
4,6-bis(3,4-dihydroxyphenyl)-3-cyano-2-methylpyridine (compound 8; DC-0008).
[0113] To a stirred solution of
DC-0008B (0.10 g) in dry CH
2Cl
2 (25 ml) under nitrogen, was slowly added boron tribromide (0.2 ml) then stirring
continued for a further 2 hours. Methanol (50 ml) was added carefully, then the solvent
evaporated in vacuo to a volume of 1 ml; this was then repeated 2 more times. The
product was recrystallized from methanol/acetone to give pure
DC-0008 as small yellow crystals (64 mg, 69%).
1H-NMR ((CD
3)
2CO) 8.19 (1H, s), 7.86 (1H, d, J 2Hz), 7.75 (1H, dd, J 2, 8Hz), 7.58 (1H, d, J 2Hz),
7.45 (1H, dd, J 2, 8 Hz), 7.16 (1H, d, J 8Hz), 7.13 (1H, d, J 8Hz), and 2.73 (3H,
s).
M/z 335 ((M +1)
+, 100%)
HPLC (method 2) 31.8 minutes.
Bis (3,4-methylenedioxy)chalcone (compound 6B; DC-0006B)
[0114]
[0115] A mixture of piperonal (460 mg) and 3,4-methylenedioxyacetophenone (500 mg) in ethanol
(20 ml) was treated with 1M NaOH solution (4 ml), then the mixture was stirred overnight.
The pale yellow crystalline solid was filtered off, washed with water then cold aqueous
ethanol and dried to give pure bis(3,4-methylenedioxy)chalcone
DC-0006B (476 mg, 53%).
1H-NMR (CDCl
3) 7.72 (1H, d, J 16Hz), 7.64 (1H, dd, J 2, 8Hz), 7.52 (1H, d, J 2Hz), 7.33 (1H, d,
J 16Hz), 7.16 (1H, d, J 2Hz), 7.12 (1H, dd, J 2, 8Hz), 6.89 (1H, d, J 8Hz), 6.84 (1H,
d, J 8Hz), 6.06 (2H, s) and 6.03 (2H, s).
M/z 297 ((M+1)
+, 100%).
Comparative Example 5: 1,4-bis(3,4-dihydroxybenzyl) piperazine (compound 9; DC-0009)
Method 1-via methylenedioxy-protected compounds
[0116]
1,4-bis-(3,4-methylenedioxybenzyl) piperazine (DC-0009B)
[0117] To a solution of piperazine (207 mg) in dry DMF (5 ml) under nitrogen was added sodium
hydride (80% w/w in oil, 250 mg), followed by 3,4-methylenedioxybenzylchloride (0.90
g) and the mixture stirred at room temperature overnight. Aqueous NaOH (50 ml, 1M)
was added slowly, then saturated NaCl solution (50 ml) and the product extracted with
dichloromethane (2 x 100 ml). The organic layer was washed with water (2 x 100 ml),
dried and evaporated in vacuo to give a white solid. Column chromatography eluting
with increasing proportions of ether in dichloromethane gave pure
DC-0009B (0.68 g, 80%) as a white powder.
1H NMR (CDCl
3) 6.85 (2H, s), 6.70 (4H, s), 5.94 (4H, s), 3.42 (4H, s) and 2.45 (8H, bs). M/z 355
((M+1)
+, 100%).
1,4-bis-(3,4-dihydroxybenzyl) piperazine (DC-0009)
[0118] To a stirred solution of
DC-0009B (200 mg) in dry CH
2Cl
2 (25 ml) under nitrogen, was slowly added boron tribromide (0.6 ml) then stirring
continued for a further 30 minutes. Methanol (50 ml) was added carefully, then the
solvent evaporated in vacuo to a volume of 1 ml, and this addition and evaporation
was repeated twice more. Purification by column chromatography over silica gel eluting
with 20% methanol in chloroform gave a fraction containing crude product
DC-0009 (51 mg, 27%) as a white powder.
1H NMR (CD
3)
2CO) 6.88 (2H, d, J 2Hz), 6.78 (2H, d, J 8Hz), 6.67 (2H, dd, J 2, 8Hz), 3.36 (4H, s)
and 2.50 (8H, bs).
13C NMR (CD
3)
2CO) 146.50,145.85, 131.17, 122.15, 117.78, 116.44, 63.72 and 54.23. M/z 331 ((M+H)
+, 100%).
HPLC (Method 2) 3.79, 3.22 minutes for the mono and di protonated forms.
Method 2 - via methoxy-protected compounds
[0119]
3,4-Dimethoxybenzyl chloride
[0120] 3,4-dimethoxybenzyl alcohol (20 g, 119 mmol) was dissolved in toluene (60 ml) and
cooled to 0°C. Thionyl chloride (7.48 g, 61.4 mmol) was added dropwise to the cooled
solution of the alcohol over a period of 30 minutes, and the reaction was maintained
at 0°C for an additional 30 minutes. The reaction was quenched by pouring onto an
ice/water mix (100 ml), and the organic phase was separated. The aqueous phase was
then extracted into toluene (2 x 20 ml) and the combined toluene solution was dried
over anhydrous sodium sulfate. The toluene was removed at reduced pressure to afford
an oil which solidified upon standing, with a yield of 21 g. The material was characterized
as a single spot by thin layer chromatography (TLC).
1,4-Bis(3,4-dimethoxybenzyl)piperazine
[0121] 3,4-dimethoxybenzyl chloride (10 g, 53.6 mmol) was combined with piperazine (2.3
g, 26.8 mmol) in anhydrous DMF (30 ml) and heated with stirring under nitrogen for
8 hours at 95-100°C. The cooled reaction mixture was diluted with water (100 ml) and
acidified to pH 1 with concentrated hydrochloric acid. The white precipitate was collected
by filtration and washed with water (50 ml). The solid was resuspended in water (50
ml) and the pH adjusted to >9 by the dropwise addition of sodium hydroxide solution
(50% NaOH in water). The resultant white solid was collected by filtration and dried
under vacuum at 50°C, yield 10 g.
1,4-Bis(3,4-dihydroxybenzyl)piperazine (DC-0009)
[0122] 1,4-Bis(3,4-dimethoxybenzyl)piperazine (5 g, 12.95 mmol) was combined with hydrobromic
acid (50 ml of 48% w/w solution in water) and the solution heated slowly over 1 hour
to 145°C. Reaction was maintained at 145°C for 12 h at which time TLC revealed disappearance
of starting material. The cooled solution was diluted with water (200 ml), carefully
neutralized with saturated aqueous sodium hydrogen carbonate, and ethyl acetate (100
ml) added. The crude aqueous solvent mixture was filtered through Celite and the ethyl
acetate layer separated. The aqueous layer was extracted with ethyl acetate (2 x 50
ml), and the combined extracts washed with water (50 ml), and dried (Na
2SO
4). The solvent was removed under reduced pressure and the residue recrystallized from
toluene and methyl ethyl ketone to afford the product,
DC-0009, 100 mg (98%, pure by HPLC analysis).
Comparative Example 6: N,N'-bis(3,4-dihydroxybenzyl)-trans-1,2-diaminocyclohexane (compound 12; DC-0012)
[0123]
N,N'-bis(3,4-methylenedioxybenzyl)-trans-1,2-diaminocyclohexane (compound 12B; DC-0012B)
[0124] To a solution of piperonal (0.8 g, 5.3 mmol) and 1,2-diaminocyclohexane (0.296 g,
2.6 mmol) in dry methanol (25 ml) was added sodium cyanoborohydride (0.38 g, 6 mmol)
and the mixture stirred for 48 h. The mixture was filtered and the solvents removed
in vacuo to give the crude product. Crystallization from methanol gave
DC-0012B as an off-white crystalline solid (0.298 g, 30%).
1H-NMR(CDCl
3) 6.83 (2H, s), 6.75 (4H, s), 5.94 (4H, m), 3.80 (2H, d, J 13Hz), 3.56 (2H, d, J 13Hz),
2.22 (2H, m), 2.18 (2H, m), 1.74 (4H, m), 1.22 (2H, m) and 1.02 (2H, m).
N,N'-bis(3,4-dihydroxybenzyl)-trans-1,2-diaminocyclohexane (compound 12; DC-0012)
[0125] To a stirred solution of
DC-0012B (0.25 g) in dry CH
2Cl
2 (25 ml) under nitrogen, was slowly added boron tribromide (0.31 ml), then stirring
was continued for a further 4 hours. Methanol (100 ml) was added carefully, then the
solvent evaporated in vacuo to a volume of 1 ml; this addition and evaporation was
then repeated twice more, and then water (2 ml) was added and the product lyophilized
to give
DC-0012 as a pale brown solid (150 mg, 64%).
1H-NMR(D
2O) 6.88 (2H, br s), 6.84 (2H, d, J 8Hz), 6.76 (2H, br d, J 8Hz), 4.20 (2H, d, J 13
Hz), 3.98 (2H, d, J 13 Hz), 3.41 (2H, br s), 2.24 (2H, br s),1.74 (2H, br s), 1.63
(2H, br s) and 1.40 (2H, br s). M/z 359 ((M+1)
+, 100%).
HPLC (Method 2) 8.2 minutes.
Comparative Example 7: 2,4-bis(3,4-dihydroxybenzyl)-3-tropinone (compound 19; DC-0019)
[0126]
[0127] A mixture of tropinone (418 mg, 3 mmol) and 3,4-methylenedioxybenzaldehyde (900 mg,
6 mmol) in ethanol (20 ml) was treated with 1M NaOH solution (4 ml), and then the
mixture was stirred overnight. The yellow crystalline solid was filtered off, washed
with water, then cold aqueous ethanol, and dried to give pure
DC-0019P (938 mg, 77%).
1H-NMR(CDCl
3) 7.73 (2H, s), 6.88 (6H, m), 6.02 (4H, s), 4.39 (2H, m), 2.60 (2H, m), 2.31 (3H,
s) and 1.98 (2H, q, J 8Hz).
M/z 404 ((M+1)
+, 100%).
2,4-bis(3,4-methylenedioxybenzyl)-3-tropinone (compound 19B; DC-0019B)
[0128] A mixture
of DC-0019P (500 mg, 1.24 mmol) and 10% Pd/C (100 mg) in ethyl acetate (50 ml) was stirred overnight
under an atmosphere of hydrogen. The mixture was filtered through Celite and evaporated
in vacuo. Crystallization of the residue from dichloromethane/ether gave pure
DC-0019B (366 mg, 72%) as a white crystalline solid.
1H-NMR(CDCl
3) 6.69 (2H, d, J 8Hz), 6.61 (2H, d, J 2Hz), 6.58 (2H, dd, J 2, 8Hz), 5.90 (4H, s),
3.17 (4H, m), 2.86 (2H, m), 2.36 (3H, s), 2.24 (2H, dd, J 8, 12Hz), 1.83 (2H, m) and
1.60 (2H, q, J 8Hz).
M/z 408 ((M+1)
+, 100%).
2,4-bis(3,4-dihydroxybenzyl)-3-tropinone (compound 19; DC-0019)
[0129] To a stirred solution of
DC-0019B (0.10 g) in dry CH
2Cl
2 (25 ml) under nitrogen, was slowly added boron tribromide (0.2 ml) then stirring
continued for a further 2 hours. Methanol (50 ml) was added carefully, then the solvent
evaporated in vacuo to a volume of 1 ml, this was repeated 2 more times. The product
was crystallized from methanol to give pure
DC-0019 (42 mg, 45%) as a white solid.
1H-NMR (D
2O) 6.75 (2H, d, J 8Hz), 6.68 (2H, d, J 2Hz), 6.59 (2H, dd, J 2, 8Hz), 3.84 (2H, bs),
3.31 (4H, s), 3.07 (2H, dd, 6, 14Hz), 2.82 (3H, s), 2.37 (dd, J 8, 14Hz) and 2.05
(2H, d 8Hz). M/z 384 ((M+1)
+, 100%).
HPLC (method 2) 30.9 minutes.
Comparative Example 8: α-(3,4-Dihydroxybenzamido)-3,4-dihydroxycinnamic acid 3,4-dihydroxybenzylamide
(compound 21; DC-0021)
[0130]
2-(3,4-methylenedioxyphenyl)-4-(3,4-methylenedioxybenzylamino)methylene-4H-oxazol-5-one (DC-0021P)
[0131] DC-0021P is also referred to as
DC-0022B, and is commercially available. It was prepared from (3,4-methylenedioxybenzoyl)aminoacetic
acid [3,4-methylenedioxyhippuric acid] (prepared by the method of
Acheson et al., J. Chem. Soc. Abstracts, 1960:3457-3461, from 3,4-methylenedioxybenzoic acid), by reaction with piperonaldehyde using the
method described by
Van der Eycken et al., Tet. Lett., 30(29):3873-3876, 1989.
1H-NMR (CDCl
3) 8.09 (1H, d, J 2Hz), 7.75 (1H, dd, J 2, 8Hz), 7.62 (1H, d, J 2Hz), 7.45 (1H, dd,
J2, 8Hz), 7.12 (1H, s), 6.94 (1H ,d, J 8Hz), 6.90 (1H, d, J 8Hz), 6.11(2H, s) and
6.08 (2H, s).
m/z 338 (M+H)
+.
α-(3,4-methylenedioxybenzamido)-3,4-methylenedioxycinnamic acid 3,4-methylenedioxybenzyl-amide
(compound 21B; DC-0021B)
[0132] A mixture of
DC-0021P (250 mg, 0.74 mmol) and 3,4-methylenedioxybenzylamine (0.112 g, 0.74 mmol) in acetic
add (glacial, 3 ml) were heated together under reflux for 30 minutes. The reaction
was quenched with ethyl acetate, washing with sodium bicarbonate, dried and evaporated
in vacuo to give the crude product. Purification by column chromatography, eluting
with hexane/ethyl acetate (50/50), followed by recrystallization from ethanol/water
gave pure
DC-0021B (218 mg, 60%).
1H-NMR ((CD
3)
2CO) 9.09 (1H, bs), 8.06 (1H, bt, J 7Hz), 7.70 (1H, dd, J 2, 8Hz), 7.56 (1H, d, J 2Hz),
7.37 (1H, s), 7.16 (1H, d, J 2Hz), 7.08 (1H, dd, J 2, 8Hz), 7.00 (1H, d, J 8Hz), 6.94
(1H, d, J 2Hz), 6.86 (1H, d, J 8 Hz), 6.84 (1H, dd, J2, 8Hz), 6.77 (1H, d, J 8Hz),
6.14 (2H, s), 6.02 (2H, s), 5.98 (2H, s) and 4.43 (2H, d, J 7Hz).
M/z 489 ((M+1)
+, 100%).
α-(3,4-dihydroxybenzamido)-3,4-dihydroxycinnamic acid 3,4-dihydroxybenzylamide (compound
21; DC-0021)
[0133] To a stirred solution
of DC-0021B (85 mg) in dry CH
2Cl
2 (20 ml) under nitrogen, was slowly added boron tribromide (0.2 ml) then stirring
continued for a further 2 hours. Methanol (50 ml) was added carefully, then the solvent
evaporated in vacuo to a volume of 1 ml; this was repeated 2 more times. Purification
by column chromatography over silica gel eluting with 20% methanol in chloroform gave
pure
DC-0021 as a pale yellow solid (42 mg, 53%).
1H-NMR ((CD
3)
2CO) 7.75 (1H, d, J 2Hz), 7.63 (1H, dd, J 2, 8Hz), 7.50 (1H, s), 7.34 (1H, d, J 2Hz),
7.12 (1H, dd, J 2, 8 Hz), 7.00-7.04 (2H, m), 6.91 (1H, d, J 8Hz), 6.80 - 6.85 (2H,
m) and 4.68 (2H, s). M/z 451 ((M-1)
+, 100%).
HPLC (method 2) 27.1 minutes.
Comparative Example 9: 1,4-bis(3,4-dihydroxybenzoyl)piperazine (compound 23; DC-0023)
[0134]
1,4-bis(3,4-methylenedioxybenzoyl)piperazine (compound 23B; DC-0023B)
[0135] A suspension of piperonylic acid (0.5 g) in thionyl chloride (15 ml) was refluxed
for 1 h under nitrogen, when a clear solution had been formed. The solvents were removed
in vacuo to give the acid chloride as a white solid. The solid was dissolved in dry
dichloromethane (7 ml) and added dropwise to a stirred solution of piperazine (0.13
g) in dry dichloromethane (20 ml) containing pyridine (0.5 ml). The mixture was refluxed
for 30 minutes, diluted with more dichloromethane (50 ml), then washed with aqueous
HCl (1M, 50 ml) followed by aqueous NaOH (1M, 50 ml), dried and evaporated in vacuo
to give the crude product Crystallization from methanol/water gave DC-0023B as a white
solid (532 mg, 92%).
1H-NMR (CDCl
3) 6.80 - 6.96 (6H, m), 6.00 (4H, s), and 3.62 (8H, bs).
1,4-bis(3,4-dihydroxybenzoyl)piperazine (compound 23; DC-0023)
[0136] To a stirred solution
of DC-0023B (0.20 g) in dry CH
2Cl
2 (25 ml) under nitrogen, was slowly added boron tribromide (0.4 ml) then stirring
continued for a further 2 hours. Methanol (50 ml) was added carefully, then the solvent
evaporated in vacuo to a volume of 1 ml, this was repeated 2 more times. The product
was crystallized from methanol/dichloromethane to give pure
DC-0023 (141 mg, 75%) as a white solid.
1H-NMR (CD
3OD) 6.88 (2H, s), 6.81 (4H, s) and 3.66 (8H, s).
M/z 357 ((M-H)
+, 100%).
Comparative Example 10: N,N'-bis(3,4-dihydroxybenzoyl)-trans-1,2-diaminocyclohexane (compound 26; DC-0026)
[0137]
N,N'-bis(3,4-methylenedioxybenzoyl)-trans-1,2-diaminocyclohexane (compound 26B; DC-0026B)
[0138] A suspension of piperonylic acid (0.5 g) in thionyl chloride (15 ml) was refluxed
for 1 h under nitrogen, when a clear solution had been formed. The solvents were removed
in vacuo to give the acid chloride as a white solid. The solid was dissolved in dry
dichloromethane (7 ml) and added dropwise to a stirred solution of
trans-1,2-diaminocyclohexane (0.17 g) in dry dichloromethane (20 ml) containing pyridine
(0.5 ml). The mixture was refluxed for 30 minutes, diluted with more dichloromethane
(50 ml), then washed with aqueous HCl (1M, 50 ml), followed by aqueous NaOH (1M, 50
ml), dried and evaporated in vacuo to give the crude product Crystallization from
methanol/water gave
DC-0026B as a white solid (544 mg, 94%).
1H-NMR (CDCl
3) 7.27 (2H, m), 6.77 (2H, d,J 8Hz), 6.67 (2H, bs), 5.98 (4H, s), 3.92 (2H, bs), 2.20
(2H, bd), 1.80 (2H, bs) and 1.38 (4H, bm).
N,N'-bis(3,4-dihydroxybenzoyl)-trans-1,2-diaminocyclohexane (compound 26; DC-0026)
[0139] To a stirred solution of
DC-0026B (0.20 g) in dry CH
2Cl
2 (25 ml) under nitrogen, was slowly added boron tribromide (0.4 ml) then stirring
continued for a further 2 hours. Methanol (50 ml) was added carefully, then the solvent
evaporated in vacuo to a volume of 1 ml, this addition and evaporation was repeated
twice more. The product was crystallized from methanol/dichloromethane to give pure
DC-0026 (161 mg, 86%) as a white solid.
1H-NMR (CD
3OD) 7.18 (2H, s), 7.11 (2H, d,J 8Hz), 6.73 (2H, d,J 8Hz), 3.89 (2H, m), 2.06 (2H,
m), 1.83 (2H, m) and 1.44 (2H, m).
M/z 385 ((M -H)
+, 100%).
HPLC (Method 1) 30.9 minutes.
Example 11: 3,4-dihydroxybenzoic acid 3,4-dihydroxyanilide (compound 51; DC-0051)
Method 1- via methylenedioxy-protected compounds
[0140]
3,4-methylenedioxybenzoic acid 3,4-methylenedioxyanilide (compound 51; DC-0051B)
[0141] To a solution of piperonylic acid (500 mg, 3 mmol) in dry CH
2Cl
2 (25 ml) under nitrogen, was added oxalyl chloride (573 mg, 4.5 mmol) with three drops
of dry DMF, and the mixture was stirred for 1 hour. Solvents were removed in vacuo
giving the acid chloride as a white solid. To a solution of the acid chloride in dry
CH
2Cl
2 (50 ml) under nitrogen, cooled to 0°C, was added dropwise, a solution made up of
3,4-(methylenedioxy)aniline (498 mg, 30.1 mmol) and pyridine (0.5 ml) in CH
2Cl
2 (5 ml). The reaction mixture was stirred for 30 minutes at room temperature, then
diluted by the addition of CH
2Cl
2 (100 ml), washed with aqueous HCl (50 ml, 10%) and sodium bicarbonate solution (50
ml) then dried. Solvents were removed in vacuo to give the crude product as a brown
crystalline material. Recrystallization from aqueous ethanol gave
DC-0051B as small silvery crystals (0.516g, 60%).
1H-NMR(CDCl
3) 7.60 (1H, br s), 7.35 (3H, m), 6.88 (2H, m), 6.78 (1H, d,J 9Hz), 6.06 (2H, s) and
5.98 (2H, s).
3,4-dihydroxybenzoic acid 3,4-dihydroxyanilide (compound 51; DC-0051)
[0142] To a solution of
DC-0051B (100 mg) in dry CH
2Cl
2 (25 ml) under nitrogen was added BBr
3 (0.2 ml) and the mixture was stirred for 6 hours. After stirring, aqueous 3M HCl
(25 ml) was carefully added to the reaction mixture. The product was extracted into
EtOAc (200 ml), dried and evaporated in vacuo to give the crude product Purification
by column chromatography (Silica: Hexane/EtOAc 30:70) gave
DC-0051 as an off-white solid (71 mg, 77%).
1H-NMR(CD
3OD) 7.60 (1H, br s), 7.38 (1H, d,J 2Hz), 7.33 (1H, dd,J 2, 8 Hz), 7.21 (1H, d,J 2Hz),
6.89 (1H, dd,J 2, 8Hz), 6.86 (1H, d,J 8Hz) and 6.76 (1H, d,J 8Hz).
M/z 262 ((M+1)
+, 100%)
HPLC (method 2) 15.1 minutes.
Method 2 - via benzyloxy- and methoxymethoxy-protected compounds:
[0143]
3,4-dibenzyloxybenzoyl chloride
[0144] 3,4-dibenzyloxybenzoic acid (3.1 g. 9.3 mmol) was combined with pyridine (5 drops,
catalytic) and thionyl chloride (15 ml, 205 mmol). The solution was heated at reflux
for 4 h, cooled, and excess thionyl chloride removed under reduced pressure. The crude
product was dissolved in benzene (50 ml), and stripped of solvent under vacuum. The
benzoyl chloride (theoretical yield 3.4 g) was then dissolved in dichloromethane and
used directly in the next step.
3,4-dibenzyloxybenzoic acid 3,4-di(methoxymethoxy)anilide
[0145] 3,4-di(methoxymethoxy)aniline (0.484 g, 2.2 mmol) was dissolved in dichloromethane
(5 ml) and pyridine (3 ml) and cooled to -5°C, while stirring under nitrogen. A solution
of 3,4-dibenzyloxybenzoyl chloride in dichloromethane (0.8 g, 2.2 mmol of acid chloride)
was added dropwise over 30 minutes. The reaction was allowed to stir at 0°C for 30
minutes then warmed to room temperature over 30 minutes. The reaction was diluted
with dichloromethane (100 ml), washed with aqueous citric acid (3 x 300 ml of a 2%
w/v solution), aqueous sodium hydroxide (2 x 35 ml of a 2% w/v solution) and dried
(Na
2SO
4). Removal of the solvent under reduced pressure afforded a solid, 0.97 g. The crude
product was triturated with warm methanol (10 ml) and filtered to afford the desired
product, 0.5 g.
3,4-dihydroxybenzoic acid 3,4-di(methoxymethoxy)anilide
[0146] 3,4-dibenzyloxybenzoic acid 3,4-di(methoxymethoxy)benzanilide (0.2 g, 0.4 mmol) was
combined with ethanol (10 ml), and palladium on charcoal (40 mg of 10% Pd/C). The
reaction was heated to reflux with stirring under nitrogen, and ammonium formate (0.8g,
12.7 mmol) was added portion wise over 15 min and then held at reflux for two hours.
The cooled reaction solution was filtered to remove the catalyst and concentrated
under reduced pressure to afford the crude product, 0.13 g.
3,4-dihydroxybenzoic acid 3,4-dihydroayanilide (compound 51; DC-0051)
[0147] 3,4-dihydroxybenzoic acid 3,4-di(methoxymethoxy)benzanilide (0.17 g, 0.49 mmol) was
combined with a 25% solution of hydrogen chloride in isopropyl alcohol (15 ml) and
water (1 ml). The reaction was stirred at room temperature for 1h and the solvent
removed under reduced pressure. Trituration with diethyl ether (5 ml) afforded DC-0051
as a solid which was dried under vacuum at 30°C, yield 60 mg.
Example 12: 3,4-dihydroxybenzoic acid 3,4-dihydroxybenzylamide (compound 52; DC-0052)
[0148]
3,4-methylenedioxybenzoic acid 3,4-methylenedioxybenzylamide (compound 52B; DC-0052B)
[0149] A suspension of piperonylic acid (0.5.g) in thionyl chloride (15 ml) was refluxed
for 1 h under nitrogen, when a clear solution had been formed. The solvents were removed
in vacuo to give the acid chloride as a white solid. The solid was dissolved in dry
dichloromethane (7 ml) and added dropwise to a stirred solution of piperonylamine
(0.45 g) in dry dichloromethane (20 ml) containing pyridine (0.5 ml). The mixture
was refluxed for 30 minutes, diluted with more dichloromethane (50 ml), then washed
with aqueous HCl (1M, 50 ml) followed by aqueous NaOH (1M, 50 ml), dried and evaporated
in vacuo to give the crude product. Crystallization from methanol/water gave
DC-0052B as a white solid (733 mg, 79%).
1H-NMR (CDCl
3) 7.27 (2H, m), 6.79 (4H, m), 6.01 (2H, s), 5.94 (2H, s) and 4.51 (2H, d,J 5Hz).
3,4-dihydroxybenzoic acid 3,4-dihydroxybenzylamide (compound 52; DC-0052)
[0150] To a stirred solution of
DC-0052B (0.20 g) in dry CH
2Cl
2 (25 ml) under nitrogen, was slowly added boron tribromide (0.4 ml) then stirring
continued for a further 2 hours. Methanol (50 ml) was added carefully, then the solvent
evaporated in vacuo to a volume of 1 ml; this was then repeated 2 more times. The
product was crystallized from methanol/dichloromethane to give pure
DC-0052 (65 mg, 35%) as a white solid.
1H-NMR (CD
3OD) 7.29 (2H, s), 7.22 (2H, d,J 8Hz), 6.78 (4H, m), 6.67 (4H, m) and 4.38 (4H, d,J
5Hz).
M/z 274 ((M -H)
+, 100%)
HPLC (Method 1) 10.4 minutes.
Example 13: 3-(3,4-dihydroxyphenyl)propionic acid 3,4-dihydroxyanilide (compound 57;
DC-0057)
[0151]
3-(3,4-methylenedioxyphenyl) propionic acid 3,4-methylenedioxyanilide (compound 57B;
DC-0057B)
[0152] To a solution of 3,4-(methylenedioxy)dihydrocinnamic acid (0.4 g) in dry CH
2Cl
2 (25 ml) under nitrogen, was added oxalyl chloride (0.5 ml) with three drops of dry
DMF and the mixture stirred for 1 hour. Solvents were removed in vacuo giving the
acid chloride as a yellow solid. To a solution of the acid chloride in dry CH
2Cl
2 (50 ml) under nitrogen, cooled to 0°C, was added dropwise, a solution of 3,4-(methylenedioxy)aniline
(0.35 g) and pyridine (0.2 ml) in CH
2Cl
2 (5 ml). The reaction mixture was stirred for 30 minutes at room temperature, diluted
with CH
2Cl
2 (100 ml), washed with aqueous HCl (100 ml, 10%) and sodium bicarbonate solution (100
ml) then dried and evaporated in vacuo to give
DC-0057B as a dark brown powder (0.549g, 85%).
1H-NMR (CDCl
3) 7.15 (1H, d,J 2Hz), 6.86 (1H, bs), 6.60 - 6.75 (5H, m), 5.93 (2H, s), 5.92 (2H,
s), 2.95 (2H, t,J 4Hz) and 2.57 (2H, t,J 4Hz).
3-(3,4-dihydroxyphenyl)propionic acid 3,4-dihydroxyanilide (compound 57; DC-0057)
[0153] To a stirred solution of
DC-0057B (0.20 g) in dry CH
2Cl
2 (25 ml) under nitrogen, was slowly added boron tribromide (0.4 ml), then stirring
was continued for a further 2 hours. Methanol (50 ml) was added carefully, then the
solvent evaporated in vacuo to a volume of 1 ml, this was repeated 2 more times to
give pure
DC-0057 as a brown solid (143 mg, 77%).
1H-NMR ((CD
3)
2CO) 7.31 (1H, s), 6.98 (3H, m), 6.84 (1H, d,J 8Hz), 6.78 (1H, dd,J 2,8Hz), 3.24 (2H,
m) and 3.16 (2H, m).
M/z 370, 368 (M+HBr)
+, 288 ((M -H)
+,100%)
HPLC (Method 2) 20.6 minutes.
Example 14: 3-(3,4-dihydroxyphenyl)propionic acid 3,4-dihydroxybenzylamide (compound
58; DC-0058)
[0154]
3-(3,4-methylenedioxyphenyl)propionic acid 3,4-methylenedioxybenzylamide (compound
58B; DC-0058B)
[0155] To a solution of 3,4-methylenedioxydihydrocinnamic acid (0.4 g) in dry CH
2Cl
2 (25 ml) under nitrogen, was added oxalyl chloride (0.5 ml) with three drops of dry
DMF and the mixture was stirred for 1 hour. Solvents were removed in vacuo giving
the acid chloride as a yellow solid. To a solution of the acid chloride in dry CH
2Cl
2 (50 ml) under nitrogen, cooled to 0°C, was added dropwise, a solution of 3,4-(methylenedioxy)benzylamine
(0.35 g) and pyridine (0.2 ml) in CH
2Cl
2 (5 ml). The reaction mixture was stirred for 30 minutes at room temperature, diluted
with CH
2Cl
2 (100 ml), washed with aqueous HCl (100 ml; 10%) and sodium bicarbonate solution (100
ml) then dried and evaporated in vacuo to give
DC-0058B as an off white powder (0.536g, 80%).
3-(3,4-dihydroxyphenyl)propionic acid 3,4-dihydroxybenzylamide (compound 58; DC-0058)
[0156] To a stirred solution of
DC-0058B (0.20 g) in dry CH
2Cl
2 (25 ml) under nitrogen, was slowly added boron tribromide (0.4 ml), then stirring
was continued for a further 2 hours. Methanol (50 ml) was added carefully, then the
solvent evaporated in vacuo to a volume of 1 ml; this was repeated 2 more times to
give pure
DC-0058 as a brown solid (143 mg, 77%).
1H-NMR ((CD
3)
2CO) 9.62 (1H, bs), 6.95 (1H, d,J 2Hz), 6.91 (1H, d,J 2Hz), 6.88 (1H, d,J 8Hz), 6.83
(1H, d,J 8Hz), 6.67 (2H, m), 6.35 (4H, bs) 4.47 (2H, s) and 3.00 (4H, m).
M/z 302 ((M -H)
+, 100%)
HPLC (Method 2) 19.4 minutes.
Example 15: 3,4-dihydroxycinnamic acid 3,4-dihydroxybenzylamide (compound 61; DC-0061)
[0157]
3,4-methylenedioxycinnamic acid 3,4-methylenedioxybenzylamide (compound 61B; DC-0061B)
[0158] To a solution of 3,4-methylenedioxycinnamic acid (0.5 g, 2.6 mmol) in dry CH
2Cl
2 (25 ml) under nitrogen, was added oxalyl chloride (0.33 g, 2.6 mmol) with three drops
of dry DMF and the mixture was stirred for 1 hour. Solvents were removed in vacuo
giving the acid chloride as a yellow solid. To a solution of the acid chloride in
dry CH
2Cl
2 (50 ml) under nitrogen, cooled to 0°C, was added dropwise, a solution of 3,4-(methylenedioxy)benzylamine
(0.393 g, 2.6 mmol) and pyridine (0.205 g, 2.6 mmol in CH
2Cl
2 (5 ml). The reaction mixture was stirred for 30 minutes at room temperature, diluted
with CH
2Cl
2 (100 ml), washed with aqueous HCl (100 ml, 10%) and sodium bicarbonate solution (100
ml) then dried and evaporated in vacuo tb give
DC-0061B as a dull yellow powder (0.523g, 62%).
1H-NMR(CDCl
3) 7.58 (1H, d,J 16Hz), 6.98 (2H, m), 6.70 - 6.84 (4H, m), 6.22 (1H, d,J 16 Hz), 6.00
(2H, s), 5.96 (2H, s) and 4.47 (2H, d,J 6Hz).
M/z 326 ((M +1)
+, 100%)
3,4-dihydroxycinnamic acid 3,4-dihydroxybenzylamide (compound 61; DC-0061)
[0159] To a stirred solution of
DC-0061B (0.3 g, 0.94 mmol) dissolved in dry CH
2Cl
2 (25ml) was slowly added boron tribromide (1.16 g, 4.6 mmol), then stirring continued
for a further 12 hours. Dilute HCl (25 ml) was carefully added, then 200 ml of water,
and the product was extracted into ethyl acetate (2 x 100 ml), dried and evaporated
in vacuo to give the crude product. Purification by column chromatography eluting
with hexane/ethyl acetate (1:4) gave
DC-0061 as an off-white solid (36 mg, 13%).
1H-NMR ((CD
3)
2CO) 7.54 (1H, d,J 16Hz), 7.12 (1H, d,J 2Hz), 6.96 (1H, dd,J 2, 8Hz), 6.85 - 6.94 (2H,
m), 6.80 (1H, d,J 8Hz), 6.70 (1H, dd,J 2,8Hz), 6.58 (1H, d, J 16 Hz) and 4.41 (2H,
s).
M/z 300 ((M -1)
+, 100%)
HPLC (method 2) 30.0 minutes.
Comparative Example 16: Oxalic acid bis(3,4-dihydroxyanilide) (compound 63; DC-0063)
Method 1-via methylenedioxy-protected compounds
[0160]
Oxalic acid bis(3,4-methylenedioxyanilide) (compound 63B; DC-0063B)
[0161] To a solution of oxalyl chloride (165 mg, 1.3 mmol) in dry CH
2Cl
2 (50ml) under nitrogen, cooled to 0°C, was added dropwise, a solution of 3,4-(methylenedioxy)aniline
(400 mg, 2.92 mmol) and pyridine (230 mg, 2.92 mmol)- dissolved in dry CH
2Cl
2 (50 ml). The reaction mixture was stirred for further 30 min at room temperature,
then washed with dilute aqueous HCl (50 ml). The organic layer was separated, dried
and evaporated in vacuo to give
DC-0063B as a gray powder (0.351 g, 82%).
1H-NMR. (CDCl
3) 10.78 (2H, s), 7.53 (2H, d,J 2Hz), 7.39 (2H, dd,J 2,8Hz), 6.96 (2H, d,J 8Hz) and
6.06 (4H, s).
Oxalic acid bis(3,4-dihydroxyanilide) (compound 63: DC-0063)
[0162] To a stirred solution of
DC-0063B (0.3 g, 0.91 mmol) dissolved in dry CH
2Cl
2 (25 ml) was slowly added boron tribromide (1.14 g, 4.7 mmol) then stirring continued
for a further 4 hours. Dilute HCl (25 ml) was carefully added, then water (200 ml)
and the product extracted into ethyl acetate (2 x 200 ml), dried and evaporated in
vacuo to give the crude product. The crude product was dissolved in acetone (25 ml)
and filtered. The acetone was evaporated in vacuo to give
DC-0063 as an off-white solid (171 mg, 62%).
1H-NMR((CD
3)
2CO) 9.72 (2H, br s), 8.05 (2H, br s), 7.89 (2H, br s), 7.52 (2H, d, J 2Hz)), 7.20
(2H, dd, J 2, 8Hz) and 6.83 (2H, d,J 8Hz).
M/z 303 ((M-1)
+, 100%)
HPLC (method 2) 29.1 minutes.
Method 2 - via methoxymethoxy-protected compounds:
[0163]
Oxalic acid bis(3,4-di(methoxymethoxy)anilide)
[0164] 3,4- di(methoxymethoxy)aniline(1.5 g, 7 mmol) was dissolved in dichloromethane (50
ml) and cooled to 0°C, while stirring under nitrogen. Pyridine (3.75 ml, 46 mmol)
was added followed by dropwise addition of oxalyl chloride (0.4 g, 3.5 mmol) in dichloromethane
(5 ml) over 20 minutes. The reaction was stirred for a further 10 min and allowed
to warm to room temperature. The suspension was filtered. The residue was washed with
hexane (5 ml) to remove excess pyridine. The crude product was triturated with methanol
(5 ml) and filtered to afford the pure protected anilide, 420 mg.
Oxalic acid bis(3,4-dihydroxyanilide)
[0165] Oxalic acid bis(3,4-di(methoxymethoxy)anilide) (0.17g, 0.36 mmol) was combined with
a 25% solution of hydrogen chloride in isopropyl alcohol (1.7 ml). The reaction was
stirred at room temperature overnight, and the solvent was removed under reduced pressure.
Trituration with diethyl ether (5 ml) afforded
DC-0063, 60 mgs.
Example 17: Succinic acid bis(3,4-dihydroxyanilide) (compound 66; DC-0066)
. Method 1- via methylenedioxy-protected compounds
[0166]
Succinic acid bis(3,4-methylenedioxyanilide) (compound 66B; DC-0066B)
[0167] To a suspension of succinic acid (200 mg, 1.7 mmol) in dry CH
2Cl
2 (25 ml) under nitrogen was added oxalyl chloride (645 mg, 5.08 mmol) with three drops
of dry DMF, and the mixture was stirred for 1 hour. Solvents were removed in vacuo
giving the acid chloride as a yellowish solid. To a stirred solution of 3,4-(methylenedioxy)aniline
(582 mg, 4.25 mmol) and pyridine (400 mg, 5.08 mmol) in dry CH
2Cl
2 (50 ml) under nitrogen at 0°C was added drop-wise a solution of the acid chloride
in dry CH
2Cl
2 (25 ml) and stirred for a further 2 hours. The solvents were removed in vacuo to
give the crude product The crude material was resuspended in EtOAc (250 ml) then washed
with dilute aqueous HCl (2 x 150 ml), saturated sodium bicarbonate (2 x 150 ml) and
water (1 x 150 ml). The EtOAc was then removed by rotary evaporation. The product
was scooped out onto filter paper and washed with water and allowed to dry to give
DC-0066B as a white solid (514 mg, 78%).
1H-NMR(CDCl
3) 9.97 (2H, s), 7.34 (2H, d,J 2Hz)), 6.99 (2H, dd,J 2,8Hz), 6.86 (2H, d,J 8Hz), 6.00
(4H, s) and 2.63 (4H, s).
Succinic acid bis(3,4-dihydroxyanilide) (compound 66; DC-0066)
[0168] To a stirred solution of
DC-0066B (0.3 g, 0.78 mmol) in dry CH
2Cl
2 (25 ml) was slowly added BBr3 (0.978 g, 3.9 mmol) then stirring continued for a further
4 hours. Dilute HCl (25 ml) was carefully added, then 200 ml of water and the product
extracted into ethyl acetate (2 x 100 ml), dried and evaporated in vacuo to give
DC-0066 as an off white solid (97 mg, 35%).
1H-NMR ((CD
3)
2CO) 8.88 (2H, br s), 7.83 (2H, br s), 7.57 (2H, br s), 7.34 (2H, d, J 2Hz), 6.90 (2H,
dd, J 2, 8Hz), 6.71 (2H, d,J 8Hz) and 2.65 (4H, s).
M/z 331 ((M-1)
+, 100%)
HPLC (method 2) 10.6 minutes.
Method 2 - via methoxymethoxy-protected compounds:
[0169]
Succinic acid bis(3,4-di(methoxymethoxy)anilide)
[0170] 3,4-di(methoxymethoxy)aniline (1 g, 4.7 mmol) was dissolved in dichloromethane (25
ml)) and cooled to 0°C, while stirring under nitrogen. Pyridine (1 ml, 12 mmol) was
added followed by dropwise addition of succinyl chloride (0.35 g, 2.3 mmol) in dichloromethane
(10 ml) over 20 minutes. The reaction was stirred for a further 2 hours and allowed
to warm to room temperature. The suspension was filtered, and the white solid collected
washed with hexane (10 ml) and then methanol (4 ml) to afford the anilide, 350 mg.
Succinic acid bis(3,4-dihydroxyanilide) (compound 66; DC-0066)
[0171] Succinic acid bis(3,9-di(methoxymethoxy)anilide) (0.15g, 0.3 mmol) was combined with
a 25% solution of hydrogen chloride in isopropyl alcohol (1.5 ml) and water (1.5 ml).
The reaction was stirred at room temperature for 3h and the solvent was removed under
reduced pressure. Trituration with diethyl ether afforded
DC-0066 as a solid which was dried under vacuum at 30°C, yield 60 mg.
Example 18: Succinic acid bis(3,4-dihydroxybenzylamide) (compound 67; DC-0067)
Method 1- via methylenedioxy-protected compounds
[0172]
Succinic acid bis(3,4-methylenedioxybenzylamide) (compound 67B; DC-0067B)
[0173] To a solution of succinic acid (200 mg, 1.7 mmol) in dry CH
2Cl
2 (25 ml) under nitrogen, was added oxalyl chloride (645 mg, 5.1 mmol) with three drops
of dry DMF and the mixture was stirred for 1 hour. Solvents were removed in vacuo
giving the acid chloride as a yellow solid. To a solution of the acid chloride in
dry CH
2Cl
2 (50 ml) under nitrogen, cooled to 0°C, was added dropwise, a solution of 3,4-methylenedioxybenzylamine
(634 mg, 4.2 mmol) and pyridine (0.33 ml) in CH
2Cl
2 (50 ml). The reaction mixture was stirred for a further 2 hours at room temperature,
then the solvents removed in vacuo to give the crude product The crude material was
resuspended in EtOAc (250 ml) then washed with dilute aqueous HCl (2 x 150 ml), saturated
sodium bicarbonate (2 x 150ml) and water (1 x 150 ml). The EtOAc was evaporated in
vacuo. Recrystallization from ethanol and water gave
DC-0067B as white flaky crystals (275 mg, 42%).
1H-NMR (DMSO-d
6) 8.31 (2H, t,J 6Hz), 6.85 (4H, m), 6.74 (2H, dd, J 2, 8Hz), 6.01 (4H, s), 4.19 (4H,
d,J 6Hz) and 2.42 (4H, s).
Succinic acid bis(3,4-dihydroxybenzylamide) (compound 67; DC-0067)
[0174] To a stirred solution of
DC-0067B (0.25 g, 0.65 mmol) dissolved in dry CH
2Cl
2 (25 ml) was slowly added boron tribromide (0.81 g, 0.31 ml), then stirring continued
for a further 4 hours. Dilute HCl (25 ml) was carefully added, then brine (125 ml)
and the product extracted into ethyl acetate (2 x 100 ml), dried and evaporated in
vacuo to give
DC-0067 as an off-white solid (180 mg, 77%).
1H-NMR((CD
3)
2CO) 7.90 (2H, br s), 7.74 (2H, br s), 7.42 (2H, br s), 6.79 (2H, d,
J 2 Hz), 6.77 (2H, d,
J 8 Hz), 6.62 (2H, dd,
J 2, 8Hz), 4.22 (4H, d,
J 7 Hz) and 2.53 (4H, s).
M/z 359 ((M -1)
+,100%)..
HPLC (method 2) 12.3 minutes.
Method 2 - via benzyloxy-protected compounds:
[0175]
Succinic acid bis(3,4-dibenzyloxybenzylamide)
[0176] 3,4-dibenzyloxybenzylamine (1.1 g, 3.45 mmol) was dissolved in anhydrous pyridine
(8 ml) and cooled to 0°C with stirring under nitrogen. To this solution, succinyl
chloride (0.23 g, 1.42 mmol) was added dropwise over 30 minutes as a solution in dichloromethane
(50 ml), while maintaining the reaction mixture at 0°C. The reaction was allowed to
warm to room temperature and stirred for an additional 45 minutes. The reaction was
poured onto crushed ice (70 g) and the dichloromethane layer was separated. The organic
extract was washed with dilute aqueous hydrochloric acid (2 x 20 ml of 0.1M solution),
water (20 ml), and dried (Na
2SO
4). Removal of the solvent at reduced pressure afforded a crude solid, which was triturated
with methanol (5 ml) to afford after filtration the protected diamide, yield 300 mg.
Succinic acid bis(3,4-dihydroxybenzylamide) (compound 67; DC-0067)
[0177] Succinic acid bis(3,4-dibenzyloxybenzylamide) (300 mg, 0.42 mmol) was dissolved in
THF (50 ml) in a pressure bottle and warmed to 35 C to ensure dissolution of the solid.
Palladium on carbon (50 mg 10% Pd/C) was added, and the vessel was pressurized with
hydrogen (to 3 atm). The reaction was agitated for 1 hour at room temperature, whereupon
TLC revealed reaction had gone to completion. The catalyst was removed by filtration,
and the solvent removed under reduced pressure to afford
DC-0067 as a crude solid (20 mg). This material was recrystallized from toluene and methanol
to afford
DC-0067.
Comparative Example 19: Bis(3,4-dihydroxybenzyl)amine (compound 73; DC-0073)
[0178]
Bis(3,4-dimethoxybenzyl)amine
[0179] To a solution of 3,4-dimethoxybenzaldelyde (1 g, 6 mmol) in anhydrous methanol (10
ml) was added 3,4-dimethoxybenzylamine (1 g, 5.9 mmol) and the solution stirred under
nitrogen at room temperature for 3 hours. The methanol was removed under reduced pressure
to afford the crude imine, 1.9 g. The imine was dissolved in THF (10 ml) and acetic
acid (4 ml), and sodium cyanoborohydride (0.38 g, 6 mmol) was added portionwise over
30 minutes. The solution was stirred for an additional 30 minutes at room temperature,
and the solvents were removed under reduced pressure, The residue was neutralized
with saturated aqueous sodium hydrogen carbonate, and the solid crude product was
isolated by filtration, and dried under vacuum at 50°C overnight, yield 0.6 g.
Bis(3,4-dihydroxybenzyl)amine (compound 73; DC-0073)
[0180] The crude bis(3,4-dimethoxybenzyl)amine (0.6 g) was combined with hydrobromic acid
(6 ml of 48% w/w solution in water) and slowly heated with stirring, to 145°C over
1 h. The reaction was maintained at 145°C for 12 h, allowed to cool to room temperature,
and poured into water (25 ml). The reaction mixture was neutralized with saturated
aqueous sodium hydrogen carbonate, and extracted with ethyl acetate (25 ml). The organic
layer was washed into water (2 x 25 ml), dried (Na
2SO
4) and the solvent removed under reduced pressure to afford
DC-0073 as a solid, 160 mg.
Comparative Example 20: Tris(3,4-dihydroxybenzyl)amine (compound 75; DC-0075)
[0181]
Tris(3,4-methylenedioxybenzyl)amine (compound 75B; DC-0075B)
[0182] To a stirred solution of piperonal (0.9 g, 6 mmol) and ammonium acetate (0.15 g,
2 mmol) in acetonitrile (25 ml) was added sodium cyanoborohydride (0.44 g, 7 mmol)
and the mixture was stirred for 4 days. The solvent was removed in vacuo, then the
residue dissolved in dichloromethane (100 ml) and washed with sat sodium bicarbonate,
dried and the solvent removed in vacuo to give a brown gum. Purification by column
chromatography over silica gel eluting with 50% dichloromethane in hexane gave the
pure
DC-0075B as a pale brown gum (135 mg, 5%.).
1H-NMR (CDCl
3) 6.91 (3H, m), 6.73 - 6.80 (6H, m), 5,94 (6H, s) and 3.42 (2H, m)
M/z 420 ((M+1)
+, 100%).
Tris(3,4-dihydcoxybenzyl)amine (compound 75; DC-0075)
[0183] To a stirred solution of
DC-0075B (135 mg) in dry CH
2Cl
2 (20 ml) under nitrogen, was slowly added boron tribromide (0.2 ml) then stirring
continued for a further 2 hours. Methanol (50 ml) was added carefully, then the solvent
evaporated in vacuo to a volume of 1 ml, this addition and evaporation was repeated
twice more. Purification by column chromatography over silica gel eluting with 20%
methanol in chloroform gave mostly pure
DC-0075 (72 mg, 58%) as a pale brown gum. Preparative HPLC then gave the pure
DC-0075 as a white gum (26 mg, 21%).
1H-NMR(CD
3OD) 6.82- 6.86 (2H, m), 6.74 (1H, dd,J 2,8Hz) and 4.07 (2H, s).
M/z 384 ((M+1)
+, 100%).
HPLC (method 2) 12.3 minutes.
Comparative Example 21: 1,3-Bis(3,4-dihydroxyphenyl)urea (compound 76; DC-0076)
[0184]
1,3-Bis(3,4-methylenedioxyphenyl)urea (compound 67B; DC-0076B)
[0185] A solution of 3,4-methylenedioxyaniline (0.35 g) and 3,4-methylenedioxyphenyl isocyanate
(0.4 g) in benzene (25 ml) was refluxed for 1 hour. The precipitate formed was filtered,
washed with benzene then dried to give pure
DC-0076B (0.697 g, 95%) as a pale brown solid.
1H-NMR (CDCl
3/(CD
3)
2CO) 7.35 (2H, bs), 6.93 (2H, s), 6.45 (4H, s) and 5.67 (4H, s).
1,3-Bis(3,4-dihydroxyphenyl)urea (compound 76; DC-0076)
[0186] To a stirred solution of
DC-0076B (150 mg) in dry CH
2Cl
2 (20 ml) under nitrogen, was slowly added boron tribromide (0.2 ml) then stirring
continued for a further 2 hours. Methanol (50 ml) was added carefully, then the solvent
evaporated in vacuo to a volume of 1 ml, and this addition and evaporation was repeated
twice more. Purification by column chromatography over silica gel eluting with 20%
methanol in chloroform gave pure
DC-0076 (113 mg, 82%) as a pale brown solid.
1H-NMR (D
2O/(CD
3)
2CO) 7.09 (2H, d,J 2Hz), 6.76 (2H, d,J 8Hz) and 6.70 (2H, dd,J 2, 8Hz).
M/z 551 ((2M-H)
+, 100%), 275 ((M -H)
+, 85%).
HPLC (Method 2) 5.8 min.
Comparative Example 22: 1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxybenzyl)urea (DC-0077)
[0187]
1-(3,4-methylenedioxyphenyl)-3-(3,4-methylenedioxybenzyl) urea (DC-0077B)
[0188] A solution of 3,4-methylenedioxybenzylamine (0.37 g) and 3,4-methylenedioxyphenyl
isocyanate (0.4 g) in benzene (25 ml) was refluxed for 1 hour. The precipitate formed
was filtered, washed with benzene then dried to give pure
DC-0077B (0.78 g, 98%) as a pale brown solid.
1H NMR (CDCl
3) 8.42 (1H, s, NH), 7.21 (1H, d, J 2Hz), 6.88 (2H, m), 6.79 (2H, m), 6.71 (1H, dd,
J 2, 8Hz), 6.49 (1H, t,J 6Hz, NH), 6.01 (2H, s), 5.97 (2H, s) and 4.21 (2H, d,J 6Hz).
M/z 315 ((M+1)
+, 100%).
1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxybenzyl) urea (DC-0077)
[0189] To a stirred solution of
DC-0077B (200 mg) in dry CH
2Cl
2 (50 ml) under nitrogen, was slowly added boron tribromide (0.4 ml) then stirring
continued for a further 3 hours. Methanol (50 ml) was added carefully, then the solvent
evaporated in vacuo to a volume of 1 ml, this was repeated 2 more times. Purification
by column chromatography over silica gel eluting with 20% methanol in chloroform gave
a fraction containing crude product Preparative HPLC gave pure
DC-0077 (19 mg, 11%), as a pale brown solid.
1H NMR (D
2O) 6.55 - 6.80 (6H, m) and 4.12 (2H, s).
M/z 290 ((M)
+, 100%).
HPLC (method 2) 12.7 min.
Comparative Example 23: 1-(3,4-Dihydroxyphenyl)-3-(3,4-dihydroxyphenethyl)urea (compound
78; DC-0078)
[0190]
1-(3,4-methylenedioxyphenyl)-3-(3,4-methylenedioxyphenethyl) urea (compound 78B; DC-0078B)
[0191] A solution of 3,4-methylenedioxyphenylethylamine (0.25 g, 1.5 mmol) and 3,4-methylenedioxy-phenyl
isocyanate (0.25 g, 1.5 mmol) in benzene (25 ml) was refluxed for 1 hour. The precipitate
formed was filtered, washed with benzene then dried to give pure
DC-0078B (0.43 g, 85%) as a pale brown solid.
1H-NMR((CD
3)
2CO) 7.83 (1H, bs), 7.31 (1H, d,J 2Hz), 6.72 - 6.82 (5H, m), 5.99 (2H, s), 5.95 (2H,
s), 5.68 (1H, bt,J 7Hz), 3.44 (2H, q,J 7Hz), and 2.74 (2H, t,J 7Hz). M/z 327 ((M-1)
+, 100%).
1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenethyl)urea (compound 78: DC-0078)
[0192] To a stirred solution of
DC-0078B (105 mg in dry CH
2Cl
2 (20 ml) under nitrogen, was slowly added boron tribromide (0.2 ml), then stirring
continued for a further 2 hours. Methanol (50 ml) was added carefully, then the solvent
evaporated in vacuo to a volume of 1 ml; this addition and evaporation was repeated
twice more. Purification by column chromatography over silica gel eluting with 20%
methanol in chloroform gave pure DC-0078 (78 mg, 80%) as a pale brown solid.
1H-NMR ((CD
3)
2CO) 6.97 (2H, m), 6.86 - 6.89 (3H, m), 6.68 (1H, dd, J 2, 8Hz), 3.66 (2H, t, J 7Hz),
and 2.87 (2H, t, J 7Hz).
M/z 303 ((M -1)
+, 100%).
HPLC (method 2) 33.7 min.
Comparative Example 24: Dibenzo[c,f][2,7]naphthyridine-2,3,10,11-tetraol (compound
85; DC-0085).
[0193]
2,3,10,11-Tetramethoxydibenzo[c,f][2,7]naphthyridine (DC-0085P)
[0194] DC-0085P was prepared as described by Upton et al.,
J.
Pharm. Pharmacol., 50(5):475-482, 1998. Veratrole was reacted with veratric acid to give the protected
benzophenone, which was nitrated to give the dinitro compound, and this was reduced
to the diamine by treatment with tin in hydrochloric add and acetic acid. The diamine
was isolated, and then condensed with malonaldehyde bis(dimethyl acetal) to give
DC-0085P.
Dibenzo[c,f][2,7]naphthyridine-2,3,10,11-tetraol (DC-0085)
[0195] To a stirred solution of
DC-0085P (100 mg) in dry CH
2Cl
2 (20 ml) under nitrogen, was slowly added boron tribromide (0.2 ml), then stirring
continued for a further 2 hours. Methanol (50 ml) was added carefully, then the solvent
evaporated in vacuo to a volume of 1 ml, and this addition and evaporation was repeated
twice more. Purification by crystallization from methanol/chloroform gave
DC-0085 (36 mg, 38%) as an orange crystalline solid.
1H-NMR (CD
3OD) 9.63 (2H, s), 8.63 (2H, s) and 7.64 (2H, s).
M/z 296 ((M+2)
+, 100%)
HPLC (method 1) 24.3 min.
Example 25: Compounds are potent disrupters of Alzheimer's Aβ 1-42 fibrils
[0196] The compounds prepared in the preceding Examples were found mostly to be potent disruptors/
inhibitors of Alzheimer's disease β-amyloid protein (Aβ) fibrils. In a set of studies,
the efficacy of the compounds to cause a disassembly/disruption of pre-formed amyloid
fibrils of Alzheimer's disease (i.e. consisting of Aβ 1-42 fibrils) was analyzed.
Part A-Thioflavin T fluorometry data
[0198] In this study, 25 µM of pre-fibrillized Aβ 1-42 (Bachem Inc) was incubated at 37°C
for 3 days either alone, or in the presence of one of the compounds or EDTA (at Aβ:test
compound weight ratios of 1:1, 1:0.1, 1:0.01 or 1:0.001). Following 3-days of co-incubation,
50 µl of each incubation mixture was transferred into a 96-well microtiter plate containing
150 µl of distilled water and 50 µl of a Thioflavin T solution (i.e. 500 mM Thioflavin
T in 250 mM phosphate buffer, pH 6.8). The fluorescence was read at 485 nm (444 nm
excitation wavelength) using an ELISA plate fluorometer after subtraction with buffer
alone or compound alone, as blank.
[0199] The results of the 3-day incubations are presented below. For example, whereas EDTA
caused no significant inhibition of Aβ 1-42 fibrils at all concentrations tested,
the compounds all caused a dose-dependent disruption/disassembly of preformed Aβ 1-42
fibrils to some extent. The most efficacious compounds to disrupt pre-formed Aβ 1-42
fibrils appeared to be compounds # 3, 4, 21, 51, 73 and 78. For example, compound
#4 caused a significant (p<0.01) 97.4±0.40% inhibition when used at an Aβ:test compound
wt/wt ratio of 1:0.1, and a 69.4±1.17% disruption when used at an Aβ:compound wt/wt
ratio of 1:0.01. Under the same conditions (i.e. Aβ:test compound wt/wt ratio of 1:0.1),
compound #3 caused an 57.8±6.36 % disruption, compound #21 caused a 81.0±1.31% disruption,
compound #51 caused 94.9±0.24 % disruption, compound #73 caused a 70.9±3.04% disruption,
and compound #78 caused a 89.7±1.8 % disruption. This study indicated that the compounds
of this invention are potent disruptors/inhibitors of Alzheimer's disease type Aβ
fibrils, and usually exert their effects in a dose-dependent manner.
Table 1: Thioflavin T fluorometry data - disruption of Aβ 1-42 Alzheimer's fibrils
% Inhibition Aβ (result±S.D.) at Aβ:test compound wt/wt ratio given |
Test Compound # |
1:1 |
1:0.1 |
1:0.01 |
1:0.001 |
EDTA (control) |
11.3±9.67 |
0.0±7.12 |
0.0±4.88 |
0.0±3.01 |
1* |
97.3±0.23 |
64.8±1.98 |
19.2±4.31 |
0.0±3.07 |
3* |
99.5±0.10 |
57.8±6.36 |
53.1±1.67 |
5.5±1.99 |
4* |
98.5±0.77 |
97.4±0.40 |
69.4±1.17 |
26.8±4.80 |
8* |
70.8±2.57 |
65.5±0.17 |
24.7±3.51 |
4.9±2.27 |
9* |
95.1±0.13 |
34.9±1.69 |
20±10.75 |
10.6±0.93 |
12* |
99.7±0.17 |
82.0±1.13 |
10.8±21.9 |
0.0±34.9 |
19* |
99.1±0.56 |
91.1±0.66 |
46.2±2.98 |
10.8±1.38 |
21* |
98.6±0.54 |
81.0±1.31 |
48.2±8.29 |
8.9±2.13 |
23* |
46.7±4.62 |
26.2±4.37 |
16.5±4.02 |
0.0±3.72 |
26* |
37.8±5.50 |
11.7±3.67 |
0.0±2.19 |
0.0±3.24 |
51 |
99.4±0.05 |
94.9±0.24 |
55.3±5.23 |
29.0±25.2 |
52 |
93.7±0.41 |
53.6±2.42 |
12.1±0.78 |
0.0±6.67 |
57 |
88.4±2.73 |
60.2±3.12 |
19.0±6.33 |
17.7±7.43 |
58 |
94.8±1.67 |
76.0±2.57 |
33.2±5.16 |
20.5±6.27 |
61 |
100.0±0.41 |
80.1±4.76 |
16.9±1.39 |
26.0±7.51 |
63* |
85.3±0.91 |
23.6±25.75 |
57.5±10.64 |
1.6±9.47 |
66 |
100.0±0.68 |
78.3±4.17 |
42.0±2.36 |
27.1±3.51 |
67 |
98.3±2.19 |
50.9±8.32 |
34.0±14.07 |
13.7±6.05 |
73* |
99.4±0.42 |
70.9±3.04 |
28.7±10.27 |
0.0±29.43 |
75* |
99.0±0.63 |
84.4±0.94 |
31.6±4.74 |
17.0±4.20 |
76* |
99.3±1.35 |
86.5±1.18 |
40.9±3.76 |
12.2±5.98 |
78* |
100±0.78 |
89.7±1.18 |
57.8±4.63 |
22.4±5.63 |
Part B: SDS-PAGE/Western blot data
[0200] The disruption of Aβ 1-42, even in its monomeric form, was confirmed by a study involving
the use of SDS-PAGE and Western blotting methods (not shown). In this latter study,
triplicate samples of pre-fibrillized Aβ 1-42 (25 µM) was incubated at 37°C for 3
days, alone or in the presence of the compounds or EDTA. Five micrograms of each sample
was then filtered through a 0.2 µm filter. Protein recovered from the filtrate was
then loaded, and ran on a 10-20% Tris-Tricine SDS-PAGE, blotted to nitrocellulose
and detected using an Aβ-antibody (clone 6E10; Senetek). In this study, Aβ 1-42 was
detected as a ∼4 kilodalton band (i.e. monomeric Aβ) following incubation alone, or
in the presence of EDTA, at 3 days. For example, Aβ 1-42 monomers were not detected
following incubation of Aβ 1-42 with compounds 4, 19, 21, 51, 58, 66, 75, 76 and 78
suggesting that these compounds were capable of causing a disappearance of monomeric
Aβ 1-42. This study confirmed that these compounds are also capable of causing a disruption/removal
of monomeric Aβ 1-42.
Part C: Congo red binding data
[0201] In the Congo red binding assay the ability of a test compound to alter amyloid (in
this case, Aβ) binding to Congo red is quantified. In this assay, Aβ 1-42 and test
compounds were incubated for 3 days and then vacuum filtered through a 0.2 µm filter.
The amount of Aβ 1-42 retained in the filter was then quantitated following staining
of the filter with Congo red. After appropriate washing of the filter, any lowering
of the Congo red color on the filter in the presence of the test compound (compared
to the Congo red staining of the amyloid protein in the absence of the test compound)
was indicative of the test compound's ability to diminish/alter the amount of aggregated
and congophilic Aβ.
[0202] In one study, the ability of Aβ fibrils to bind Congo red in the absence or presence
of increasing amounts of the compounds or EDTA (at Aβ:test compound weight ratios
of 1:1, 1:0.1, 1:0.01 or 1:0.001) was determined. The results of 3-day incubations
are presented in Table 2 below. Whereas EDTA caused no significant inhibition of Aβ
1-42 fibril binding to Congo red at all concentrations tested, the compounds caused
a dose-dependent inhibition of Aβ binding to Congo red. For example, compound #4 caused
a significant (p<0.01) 73.0±0.90% inhibition of Congo red binding to Aβ 1-42 fibrils
when used at an Aβ:test compound wt/wt ratio of 1:1, and a significant (p<0.01) 46.8±1.28%
inhibition of Congo red binding when used at an Aβ:test compound wt/wt ratio of 1:0.1,
and a significant (p<0.01) 16.4±2.02% inhibition of Congo red binding when used at
an Aβ:test compound wt/wt ratio of 1:0.01. In another example, synthetic analog compound
#3 caused a significant (p<0.01) 91.6±5.19% inhibition of Congo red binding to Aβ
1-42 fibrils when used at an Aβ:test compound wt/wt ratio of 1:1, and a significant
(p<0.01) 35.7±3.29% inhibition of Congo red binding when used at an Aβ:test compound
wt/wt ratio of 1:0.01. This study also indicated that compounds of this invention
are potent inhibitors of Aβ fibril binding to Congo red, and usually exert their effects
in a dose-dependent manner.
Table 2: Congo red binding data
% Inhibition Aβ (result±S.D.) at Aβ:test compound wt/wt ratio given |
Test Compound # |
1:1 |
1:0.1 |
1:0.01 |
1:0.001 |
EDTA (control) |
1.1±7.02 |
3.6±8.68 |
0.0±3.91 |
7.91±3.61 |
1* |
42.4±1.58 |
8.0±1.80 |
3.9±0.66 |
0.0±3.54 |
3* |
91.6±5.19 |
35.7±3.29 |
7.4±1.51 |
1.7±4.21 |
4* |
73.0±0.90 |
46.8±1.28 |
16.4±2.02 |
2.3±1.80 |
8* |
17.7±1.86 |
9.7±0.69 |
1.1±0.96 |
0.0±3.55 |
9* |
46.8±1.50 |
10.9±2.18 |
0.0±2.15 |
3.1±3.66 |
12* |
63.0±1.63 |
20.8±2.22 |
17.9±7.33 |
4.1±6.60 |
19* |
48.1±2.00 |
22.4±2.19 |
7.4±2.20 |
0.0±1.01 |
21* |
66.2±1.26 |
33.9±1.02 |
9.3±5.68 |
3.6±0.58 |
23* |
10.7±2.84 |
2.9±0.43 |
0.0±0.72 |
12.3±6.57 |
26* |
4.5±2.03 |
0.0±1.35 |
6.1±4.26 |
0.0±2.64 |
51 |
78.6±1.49 |
46.7±1.29 |
20.5±11.48 |
6.0±11.47 |
52 |
35.4±1.28 |
12.7±2.35 |
0.0±1.29 |
0.0±3.68 |
57 |
44.8±0.77 |
14.2±1.56 |
0.1±2.09 |
0.0±4.73 |
58 |
52.2±2.65 |
21.1±3.67 |
6.6±3.49 |
2.5±4.22 |
61 |
48.9±4.69 |
24.6±10.85 |
2.0±2.89 |
0.0±4.06 |
63* |
32.5±5.66 |
8.5±8.01 |
20.1±10.35 |
0.0±1.93 |
66 |
55.9±6.83 |
27.7±11.26 |
7.7±0.19 |
0.6±6.61 |
67 |
31.5±11.25 |
13.8±11.25 |
8.2±7.08 |
0.0±4.98 |
73* |
53.4±1.84 |
22.6±3.51 |
0.6±5.04 |
0.0±15.17 |
75* |
59.2±3.23 |
12.8±0.59 |
6.8±2.55 |
2.4±2.95 |
76* |
66.6±0.68 |
27.8.±7.71 |
4.1±2.23 |
0.3±5.1 |
78* |
71.1±1.09 |
39.9±3.94 |
15.4±1.39 |
3.5±1.33 |
Part D - Circular dichroism spectroscopy data
[0203] Circular dichroism (CD) spectroscopy is a method that can be used to determine the
effects of test compounds to disrupt the secondary structure conformation of amyloid
fibrils. In one study, as described in this example, circular dichroism spectroscopy
was used to determine the effects of different cmopounds of the invention on β-sheet
conformation of Aβ 1-42 fibrils. For this study, Aβ 1-42 (Bachem Inc., Torrance, CA)
was first dissolved in a 2 mM NaOH solution, maintaining the pH of these solutions
above 10. Aβ 1-42 peptides (at 25µM), in the absence or presence of test compounds,
were made up in 150 mM NaF, 50 mM phosphate buffer, pH 7.4 with 10% trifluoroethanol.
Aβ 1-42 was then incubated at 37°C in the absence or presence of different compounds
at an Aβ:test compound wt/wt ratios of 1:0.1, 1:1 and 1:10. After 3 days of incubation,
CD spectra were recorded on a Jasco 810 spectropolarimeter (Easton, MD). All CD spectra
were collected with 0.05 cm quartz cells. Wavelength traces were scanned from 190-260
nm at 0.5 nm increments with a bandwidth of 5 nm, at a scan speed of 10 nm/minute,
a response time of 32 seconds, and a data pitch of 0.5 nm. The whole system was equilibrated
and continuously flushed with nitrogen at 10 ml/minute. For data processing, the average
of 5 replicates of "test-compound" spectra were subtracted from the average of 5 replicates
of "Aβ 1-42 + test compound" spectra to determine the effects of each test compound
on disruption of Aβ 1-42 fibrils. Ellipticity in degrees was converted to MRE ([Q];
molar residue ellipticity) using the formula [Q] = 100•Q•RMW/d•c; where Q is the ellipticity
in degrees; RMW is the average residue molecular weight (∼107 daltons for Aβ 1-42);
d is the pathlength in cm (i.e. 0.05 cm); and c is the concentration in mg/ml (i.e.
0.1 mg/ml).
Figure 1 shows some of the CD spectra generated in this study. Aβ 1-42 alone in 10%
TFE PBS buffer usually demonstrated the typical CD spectra of an amyloid protein with
significant β-sheet structure, as demonstrated by the minima observed at 218 nm. However,
in the presence of test compounds (such as the compounds #4, 12, 51 and 61 shown in
Figure 1) a marked disruption of β-sheet structure in Aβ 1-42 fibrils was evident
(with a significant increase in random coil or α-helix) as shown by the flattening
out of the minima observed at 218 nm (compare to Aβ 1-42 alone). This was usually
observed at both 3 days (as seen in Figure 1) and 7 days (not shown) following co-incubation
of Aβ 1-42 fibrils with the compounds.
Figure 2 shows the effect of compound #78 on disruption of Aβ 1-42 fibrils. As shown
in this figure, Aβ 1-42 alone demonstrates the typical CD spectra of a predominant
β-sheet structure, with a marked minima observed at 218 nm. However, in the presence
of compound #78 at 3 days, there is a marked decrease in the minima usually observed
at 218 nm (with Aβ 1-42 only), indicative of a disruption of the β-sheet structure
of Aβ 1-42 fibrils.
Figure 3 shows the dose-response effects of compounds #12, 51 and 61 on disruption
of the β-sheet structure of Aβ 1-42 fibrils. As an example, increasing concentrations
of test compounds #12, 51 and 61 (at Aβ:test compounds wt/wt ratios of 1:0.1, 1:1
and 1:10) caused a general disruption of β-sheet structure as demonstrated by the
dose-dependent decrease in the minima observed at 218 nm (when compared to the minima
at 218 nm observed with Aβ 1-42 only). Compound #51 was particularly effective when
used at an Aβ:test compound wt/wt ratio of 1:10 and was shown to completely disrupt
the β-sheet structure of Aβ 1-42 fibrils as shown by the complete flattening out of
the minima observed at 218 nm (compare to Aβ 1-42 alone) (Fig. 3).
[0204] The CD studies demonstrate that the compounds of this invention have the ability
to disrupt/disassemble the β-sheet structure characteristic of Alzheimer's Aβ fibrils.
The results of the studies also confirm the previous examples using Thioflavin T fluorometry,
SDS-PAGE/ECL, and Congo red binding type assays, that the compounds of this invention
are potent anti-amyloid agents.
Example 26: Compounds are potent disrupters of type 2 diabetes IAPP fibrils
[0205] The compounds prepared in the synthetic Examples were found also to be potent disruptors/
inhibitors of type 2 diabetes IAPP fibrils. In a set of studies, the efficacy of the
compounds to cause a disassembly/disruption of pre-formed IAPP fibrils of type 2 diabetes
was analyzed.
Part A - Thioflavin T fluorometry data
[0207] In this study, 25 µM of pre-fibrillized IAPP (Bachem Inc) was incubated at 37°C for
3 days either alone, or in the presence of one of the compounds or EDTA (at IAPP:test
compound weight ratios of 1:1, 1:0.1, 1:0.01 or 1:0.001). Following 3-days of co-incubation,
50 µl of each incubation mixture was transferred into a 96-well microtiter plate containing
150 µl of distilled water and 50 µl of a Thioflavin T solution (i.e. 500 mM Thioflavin
T in 250 mM phosphate buffer, pH 6.8). The fluorescence was read at 485 nm (444 nm
excitation wavelength) using an ELISA plate fluorometer after subtraction with buffer
alone or compound alone, as blank.
[0208] The results are presented in Table 3 below. For example, whereas EDTA caused no significant
inhibition of IAPP fibrils at all concentrations tested, the compounds all caused
a dose-dependent disruption/disassembly of pre-formed IAPP fibrils to various extents.
The most efficacious compounds to disrupt IAPP fibrils appeared to be compounds #
3, 4, 23, 63, and 78. For example, compound #3 caused a significant (p<0.01) 97.7±0.19%
inhibition when used at an IAPP:test compound ratio of 1:0.1, and a 79.9±1.47% disruption
when used at a IAPP:compound wt/wt ratio of 1:0.01. Under the same conditions (i.e.
IAPP:test compound wt/wt ratio of 1:0.1), compound #4 caused a 96.0±1.0% disruption,
compound #23 caused a 67.2±18.35% disruption, compound #63 caused a 84.2±1.16% disruption,
compound #78 caused a 92.4±0.27% disruption, and compound #26 caused a 45.9±17.73%
disruption. This study indicated that the compounds of this invention are also potent
disruptors/inhibitors of type 2 diabetes IAPP fibrils, and usually exert their effects
in a dose-dependent manner.
Table 3: Thioflavin T fluorometry data - disruption of type 2 diabetes IAPP fibrils
% Inhibition IAPP (result±S.D.) at IAPP:test compound wt/wt ratio given |
Test Compound # |
1:1 |
1:0.1 |
1:0.01 |
1:0.001 |
EDTA (control) |
4.4±9.23 |
0.1±2.59 |
0.0±5.23 |
4.2±1.05 |
1* |
99.0±0.11 |
93.0±1.27 |
57.3±0.16 |
6.4±4.40 |
3* |
100±0.20 |
97.7±0.19 |
79.9±1.47 |
30.7±6.71 |
4* |
99.7±0.23 |
96.0±0.10 |
63.2±2.09 |
17.3±4.07 |
8* |
72.8±1.77 |
67.8±1.74 |
29.6±5.97 |
11.4±12.78 |
12* |
99.9±0.19 |
86.0±0.76 |
37.5±0.76 |
13.0±10.34 |
19* |
100.0±0.24 |
94.0±0.10 |
51.7±2.98 |
16.7±10.20 |
21* |
98.5±0.06 |
85.4±0.86 |
25.8±3.61 |
5.4±15.41 |
23* |
85.2±0.55 |
67.2±18.35 |
44.3±32.47 |
27.3±45.38 |
26* |
52.5±2.44 |
45.9±17.73 |
24.6±6.77 |
3.7±4.67 |
51 |
99.9±0.11 |
96.6±1.00 |
56.6±1.69 |
11.8±6.45 |
52 |
97.9±0.19 |
86.9±3.09 |
49.2±4.47 |
16.0±8.42 |
57 |
94.1±0.46 |
73.2±1.19 |
37.3±0.78 |
1.9±5.24 |
58 |
98.1±1.04 |
87.6±1.16 |
48.8±2.05 |
8.9±6.87 |
61 |
96.8±0.47 |
83.6±1.27 |
35.4±5.68 |
0.5±6.33 |
63* |
94.9±0.65 |
84.2±1.16 |
56.2±8.77 |
19.0±0.30 |
66 |
98.5±0.06 |
94.0±2.88 |
47.6±8.16 |
11.1±5.28 |
67 |
98.6±0.22 |
81.4±6.96 |
34.8±1.87 |
16.1±12.40 |
75* |
100±0.35 |
90.0±0.27 |
43.9±5.34 |
6.0±6.46 |
76* |
99.6±1.01 |
87.5±1.89 |
41.5±6.67 |
9.0±0.32 |
78* |
99.5±0.26 |
92.4±0.27 |
58.3±1.20 |
15.3±4.73 |
Part B: Congo red binding data
[0209] In the Congo red binding assay the ability of a given test compound to alter amyloid
(in this case, IAPP) binding to Congo red is quantified. In this assay, IAPP and test
compounds were incubated for 3 days and then vacuum filtered through a 0.2 µm filter.
The amount of IAPP retained in the filter was then quantitated following staining
of the filter with Congo red. After appropriate washing of the filter, any lowering
of the Congo red color on the filter in the presence of the test compound (compared
to the Congo red staining of the amyloid protein in the absence of the test compound)
was indicative of the test compound's ability to diminish/alter the amount of aggregated
and congophilic IAPP.
[0210] In the study, the ability of IAPP fibrils to bind Congo red in the absence or presence
of increasing amounts of the compounds or EDTA (at IAPP:test compound weight ratios
of 1:1, 1:0.1, 1:0.01 or 1:0.001) was determined. The results of 3-day incubations
are presented in Table 4 below. Whereas EDTA caused no significant inhibition of IAPP
fibril binding to Congo red at all concentrations tested, the compounds usually caused
a dose-dependent inhibition of IAPP binding to Congo red. For example, compound #3
caused a significant (p<0.01) 55.5±2.68% inhibition of Congo red binding to IAPP fibrils
when used at an IAPP:test compound wt/wt ratio of 1:1, and a significant (p<0.01)
37.9±3.10% inhibition of Congo red binding when used at an IAPP:test compound wt/wt
ratio of 1:0.1. Compound #4 caused a significant (p<0.01) 68.9±1.22% inhibition of
Congo red binding to IAPP fibrils when used at an IAPP:test compound wt/wt ratio of
1:1, and a 25.4±4.68% inhibition of Congo red binding when used at a NAC:test compound
wt/wt ratio of 1:0.01. This study indicated that compounds of this invention are also
potent inhibitors of type 2 diabetes IAPP fibril binding to Congo red, and usually
exert their effects in a dose-dependent manner.
Table 4: Congo red binding data
% Inhibition IAPP (result±S.D.) at IAPP:test compound wt/wt ratio given |
Test Compound # |
1:1 |
1:0.1 |
1:0.01 |
1:0.001 |
EDTA (control) |
0.0±3.69 |
0.0±1.91 |
3.6±2.83 |
6.6±2.27 |
1* |
40.7±2.49 |
10.6±3.40 |
18.6±4.05 |
6.4±2.07 |
3* |
55.5±2.68 |
37.9±3.10 |
16.3±1.13 |
11.1±5.26 |
4* |
68.9±1.22 |
25.4±4.68 |
9.0±0.51 |
0.0±1.05 |
8* |
0.0±2.84 |
0.0±2.94 |
7.2±2.27 |
0.0±6.46 |
12* |
39.8±0.26 |
8.3±0.85 |
6.9±2.45 |
0.0±2.40 |
19* |
49.3±3.97 |
21.0±3.70 |
6.0±0.78 |
2.9±4.40 |
21* |
35.9±0.21 |
10.4±3.53 |
5.1±4.53 |
0.0±2.10 |
23* |
5.5±2.33 |
4.5±4.12 |
9.3±1.40 |
5.1±2.45 |
26* |
0.0±1.21 |
7.5±2.83 |
5.3±6.14 |
10.8±2.63 |
51 |
55.6±1.48 |
27.5±3.49 |
3.6±2.59 |
1.6±1.01 |
52 |
31.3±0.27 |
11.5±1.21 |
11.0±3.27 |
10.2±0.52 |
57 |
15.7±3.77 |
8.9±3.90 |
8.5±3.19 |
4.5±0.64 |
58 |
24.5±0.57 |
0.7±6.21 |
4.6±2.35 |
0.0±1.93 |
61 |
23.7±0.39 |
0.0±7.07 |
4.0±1.78 |
0.0±3.87 |
63* |
15.4±1.34 |
4.5±1.62 |
11.7±2.26 |
0.0±2.25 |
66 |
41.4±3.84 |
15.7±2.53 |
5.7±4.23 |
4.8±1.86 |
67 |
26.3±2.76 |
5.5±2.52 |
10.6±1.29 |
0.0±3.45 |
75* |
49.0±1.17 |
7.4±0.70 |
11.3±2.24 |
2.9±0.69 |
76* |
53.9±5.44 |
16.5±2.60 |
14.2±2.25 |
3.4±1.07 |
78* |
56.3±5.32 |
16.7±6.80 |
19.9±2.12 |
6.6±3.04 |
Example 27: Compounds are potent disrupters of Parkinson's disease NAC fibrils
[0211] The tested compounds were found also to be potent disruptors/inhibitors of Parkinson's
disease NAC fibrils. NAC is a 35-amino acid fragment of α-synuclein that has been
demonstrated to form amyloid-like fibrils when incubated at 37°C for a few days. It
is the amyloidogenic fragment of α-synuclein and is postulated to play an important
role in the pathogenesis of Parkinson's disease and other synucleinopathies. In a
set of studies, the efficacy of the compounds to cause a disassembly/disruption of
pre-formed NAC fibrils of Parkinson's disease was analyzed.
Part A - Thioflavin T fluorometry data
[0212] In one study, Thioflavin T fluorometry was used to determine the effects of compounds
#1, 3, 23, 26, 52, 63, 66, 67, and EDTA (as a negative control). In this assay, Thioflavin
T binds specifically to NAC fibrils, and this binding produces a fluorescence enhancement
at 485 nm that is directly proportional to the amount of NAC fibrils present. The
higher the fluorescence, the greater the amount of NAC fibrils present (
Naki et al, Lab. Invest. 65:104-110, 1991;
Levine III, Protein Sci. 2:404-410, 1993;
Amyloid: Int. J. Exp. Clin. Invest. 2:1-6, 1995).
[0213] In this study, 25 µM of pre-fibrillized NAC (Bachem Inc) was incubated at 37°C for
3 days either alone or in the presence of dihydroxy synthetic analog compounds #1,
3, 23, 26, 52, 63, 66, 67, or EDTA (at NAC:test compound weight ratios of 1:1, 1:0.1,
1:0.01 or 1:0.001). Following 3-days of co-incubation, 50 µl of each incubation mixture
was transferred into a 96-well microtiter plate containing 150 µl of distilled water
and 50 µl of a Thioflavin T solution (i.e. 500 mM Thioflavin T in 250 mM phosphate
buffer, pH 6.8). The fluorescence was read at 485 nm (444 nm excitation wavelength)
using an ELISA plate fluorometer after subtraction with buffer alone or compound alone,
as blank.
[0214] The results of the 3-day incubations are presented below in Table 5. For example,
whereas EDTA caused no significant inhibition of NAC fibrils at all concentrations
tested, compounds 1, 3, 52, 63, 66, and 67 all caused a dose-dependent disruption/disassembly
of pre-formed NAC fibrils to various extents. For example, compound #3 caused a significant
(p<0.01) 91.0±1.99% inhibition when used at an NAC:test compound ratio of 1:0.1, and
a 93.9±0.77% disruption when used at a NAC:compound wt/wt ratio of 1:0.01. Under the
same conditions (i.e. NAC:test compound wt/wt ratio of 1:0.1), compound #1 caused
a 99.5±0.53% disruption, compound #26 caused a 61.3±6.52% disruption, compound #52
caused a 89.2±1.49% disruption, compound #66 caused a 82.5±5.37% disruption, and compound
#67 caused a 50.0±7.03% disruption. This study indicated that compounds of this invention
are potent disruptors/ inhibitors of Parkinson's disease NAC fibrils, and usually
exert their effects in a dose-dependent manner.
Table 5: Thioflavin T fluorometry data - disruption of Parkinson's disease NAC fibrils
'
% Inhibition NAC (result±S.D.) at NAC:test compound wt/wt ratio given |
Test Compound # |
1:1 |
1:0,1 |
1:0.01 |
1:0.001 |
EDTA (control) |
20.0±11.8 |
0.0±5.87 |
0.0±10.87 |
0.0±11.6 |
1* |
100.0±1.00 |
99.5±0.53 |
68.2±2.55 |
0.0±7.14 |
3* |
98.0±1.78 |
91.0±1.99 |
93.9±0.77 |
67.3±6.37 |
23* |
58.0±8.43 |
53.3±12.02 |
35.6±9.73 |
0.0±26.42 |
26* |
70.4±3.22 |
61.3±6.52 |
56.8±4.60 |
0.0±16.88 |
52 |
99.7±1.93 |
89.2±1.49 |
79.6±6.43 |
13.8±10.49 |
63* |
45.6±31.03 |
34.5±17.15 |
33.0±1.69 |
17.3±12.57 |
66 |
98.9±0.65 |
82.5±5.37 |
43.4±3.45 |
30.5±9.55 |
67 |
97.4±1.19 |
50.0±7.03 |
30.6±5.75 |
11.9±15.98 |
Part B: Congo red binding data
[0215] In the Congo red binding assay, the ability of a given test compound to alter amyloid
(in this case, NAC) binding to Congo red is quantified. In this assay, NAC and test
compounds were incubated for 3 days and then vacuum filtered through a 0.2 µm filter.
The amount of NAC retained in the filter was then quantitated following staining of
the filter with Congo red. After appropriate washing of the filter, any lowering of
the Congo red color on the filter in the presence of the test compound (compared to
the Congo red staining of the amyloid protein in the absence of the test compound)
was indicative of the test compound's ability to diminish/alter the amount of aggregated
and congophilic NAC.
[0216] In one study, the ability of NAC fibrils to bind Congo red in the absence or presence
of increasing amounts of compounds #1, 3, 23, 26, 63, 66, 67, or EDTA (at NAC:test
compound weight ratios of 1:1, 1:0.1, 1:0.01 or 1:0.001) was determined. The results
of 3-day incubations are presented in Table 6. Whereas EDTA caused no significant
inhibition of NAC fibril binding to Congo red at all concentrations tested, the compounds
tested caused a dose-dependent inhibition of NAC binding to Congo red as demonstrated
in Table 6 below. For example, compound #3 caused a significant (p<0.01) 94.4±2.48%
inhibition of Congo red binding to NAC fibrils when used at a NAC:test compound wt/wt
ratio of 1:1, and a significant (p<0.01) 83.2±3.57% inhibition of Congo red binding
when used at a NAC:test compound wt/wt ratio of 1:0.1. In comparison, compound #1
caused a 75.4±2.96% inhibition of Congo red binding to NAC fibrils when used at a
NAC:test compound wt/wt ratio of 1:1, and an 75.9±2.48% inhibition of Congo red binding
when used at a NAC:test compound wt/wt ratio of 1:0.1. In another example, synthetic
analog compound #67 caused a significant (p<0.01) 81.2 +/- 2.87% inhibition of Congo
red binding to NAC fibrils when used at an NAC:test compound wt/wt ratio of 1:1, and
a significant (p<0.01) 47.7±8.20% inhibition of Congo red binding when used at a NAC:test
compound wt/wt ratio of 1:0.01. In another example, compound #26 caused a significant
34.4±10,19% inhibition of Congo red binding when used at a NAC:test compound ratio
of 1:1, and a 36.7%±5.59% inbibition of Congo red binding when used at a NAC:test
compound ratio of 1:0.1. This study also indicated that compounds of this invention
are also potent inhibitors of Parkinson's disease type NAC fibril binding to Congo
red, and usually exert their effects in a dose-dependent manner.
Table 6: Congo red binding data - disruption of Parkinson's disease NAC fibrils
% Inhibition NAC (result±S.D.) at NAC:test compound wt/wt ratio given |
Test Compound # |
1:1 |
1:0.1 |
1:0.01 |
1:0.001 |
EDTA (control) |
0.2±7.33 |
0.0±38.26 |
0.0±22.0 |
0.0±20.57 |
1* |
75.4±2.96 |
75.9±2.58 |
40.7±4.23 |
0.0±11:39 |
3* |
94.4±2.48 |
83.2±3.57 |
81.7±2.82 |
65.2±5.40 |
23* |
41.0±8.54 |
30.3±12.06 |
25.6±5.37 |
0.0±9.00 |
26* |
34.4±10.19 |
36.7±5.59 |
36.4±0.67 |
0.0±27.34 |
52 |
73.8±3.15 |
71.2±7.17 |
78.9±4.76 |
0.0±24.43 |
63* |
54.5±7.56 |
9.3±10.5 |
34.0±3.66 |
0.0±30.84 |
66 |
81.1±1.74 |
72.4±1.79 |
51.0±9.50 |
19.5±37.59 |
67 |
81.2±2.87 |
47.7±8.20 |
39.2±10.25 |
15.5±41.42 |
Example 28: Other bis- and tris-dihydroxyaryl compounds of the invention
[0217] Besides the 24 compounds described in detail in Examples 1 - 24, this Example describes
other bis- and tris(dihydroxyaryl) compounds that also serve as potent disruptor/inhibitors
of amyloid fibrils in Alzheimer's disease (i.e. Aβ), type 2 diabetes (i.e. IAPP),
other amyloid diseases, as well as in Parkinson's disease (i.e. α-synuclein/NAC) and
other synuclein fibril diseases. A common structural motif that is present in all
of the compounds disclosed herein is the presence of two or three dihydroxyaryl groups.
These compounds are compounds # 24, 47, 53, 54, 55, 56, 59, 60, 62, 68, 69, 70, 71,
and 72. These are also referred respectively to as 0024, DC-0047, DC-0053, DC-0054,
DC-055, DC-0056, DC-0059, DC-0060, DC-0062, DC-0068, DC-0069, DC-0070, DC-0071, and
DC-0072, respectively.
[0218] These compounds may be prepared by the methods used to produce the compounds illustrated
in Examples 1 through 23 and variations thereof easily determinable by a person of
ordinary skill in the art. Thus, for example, compound 24 : may be prepared by the
method used for compound 12, substituting N,N'-dimethylethylenediamine for the
trans-1,2-diaminocyclohexane of Example 6, and so on. A person of ordinary skill in the
art will have no difficulty, having regard to that skill and this disclosure, in preparing
the compounds illustrated above or the compounds of the formula given in claim 1.
Comparative Example 29: Methylenedioxy Analogs
[0219] A strategy for the delivery of the dihydroxyaryl compounds of this invention to improve
and/or cause more favorable metabolism and bioavailability characteristics involves
the protection of the hydroxy groups of the dihydroxyaryl compounds with methylenedioxy
groups. This strategy is exemplified in the 80 structures shown below
[0220] Methylenedioxy analogs represent intermediate hydroxy protecting structures that
are made to successfully complete the synthesis of the dihydroxyaryl compounds described
in the invention. These closed-ring compounds also tend to be more stable, and hydrophobic
(water insoluble), and less likely to be altered or degraded due to the oxidation
that could occur if hydroxyl groups were present In addition, these compounds make
good prodrugs especially for delivery to the brain due to their hydrophobic nature.
Hydrophobic compounds that are lipid soluble tend to be attractive compounds for brain
delivery since they are usually able to penetrate the blood-brain-barrier.
Comparative Example 30: Acylated compounds
[0222] Another potential strategy for the delivery of the bis- and tris-dihydroxyaryl compounds
of this invention to improve and/or cause more favorable metabolism and bioavailability
characteristics, involves methods of protecting the hydroxy groups as their pharmaceutically
acceptable esters. Ester groups replacing the hydroxy groups also tend to make the
compounds more stable, and less likely to be altered or degraded due to oxidation
of the hydroxyl groups.
[0223] The compound table below illustrates the acetyl esters of the dihydroxyaryl compounds
of Examples 1 - 23 and 28 are presented below in which the OH groups are replaced
by acetyl groups. The illustration of acetyl esters here is merely exemplary for the
class of pharmaceutically acceptable esters that are part of the compounds of this
invention and may be prepared by analogous methods. The compounds of Example 29 also
form pharmaceutically acceptable esters in the same manner, and these compounds, though
not illustrated in the compound table below, are also compounds of this invention.
[0224] These compounds are expected to be efficacious in their ability to treat amyloid
diseases and synucleinopathies once the ester linkages are cleaved (by enzymes in
the plasma or in the brain tissue), and the hydroxyl groups are regenerated. Replacement
of the hydroxyl groups with ester groups will yield prodrugs that are believed to
improve toxicity (i.e. being less toxic), metabolism (since the OH groups will be
less likely to be altered by methylation, glucuronidation and sulfation), and bioavailability.
In this prodrug concept, it is believed that the prodrug conversion takes place in
the plasma (following its protection through the gut), and closer to its appropriate
target tissue (systemic organs for the treatment of systemic amyloid diseases and/or
brain for the treatment of Alzheimer's, Parkinson's, type 2 diabetes, and other Aβ
amyloid and synuclein diseases). Enzymes in the blood and appropriate tissues are
believed to be able to cleave the ester linkages on these pharmaceutically acceptable
esters to yield the dihydroxy structures important for the observed efficacy against
Alzheimer's disease, other amyloid diseases (such as IAPP fibrils in type 2 diabetes),
and (α-synuclein/NAC fibrils, such as in Parkinson's disease, and other synucleinopathies.
[0225] The pharmaceutically acceptable esters of compounds #1 through #86 are prepared by
methods well known to persons of ordinary skill in the art, such as by reaction of
the dihydroxyaryl compounds with pharmaceutically acceptable acids, especially in
activated form (such as the acyl halides) and/or in the presence of reagents facilitating
esterification (such as an acidic catalyst) and/or under conditions favoring esterification
(such as by conducting the reaction under conditions where the water formed in the
esterification is removed, e.g. by distillation). Methods of esterification of phenolic
hydroxyl groups are well known to persons of ordinary skill in the art.
Example 31: Compositions of compounds
[0227] The compounds of this invention, as mentioned previously, are desirably administered
in the form of pharmaceutical compositions. Suitable pharmaceutical compositions,
and the method of preparing them, are well-known to persons of ordinary skill in the
art and are described in such treatises as
Remington: The Science and Practice of Pharmacy, A. Gennaro, ed., 20th edition, Lippincott,
Williams & Wilkins, Philadelphia, PA.
[0228] Representative compositions are as follows:
Oral tablet formulation
[0229] An oral tablet formulation of a compound of this invention is prepared as follows:
|
% w/w |
Compound of this invention |
10.0 |
Magnesium stearate |
0.5 |
Starch |
2.0 |
Hydroxypropylmethylcellulose |
1.0 |
Microcrystalline cellulose |
86.5 |
[0230] The ingredients are mixed to homogeneity, then granulated with the aid of water,
and the granulates dried. The granulate is then compressed into tablets sized to give
a suitable dose of the compound. The tablet is optionally coated by applying a suspension
of a film forming agent (e.g. hydroxypropylmethylcellulose), pigment (e.g. titanium
dioxide), and plasticizer (e.g. diethyl phthalate), and drying the film by evaporation
of the solvent. The film coat may comprise, for example, 2-6% of the tablet weight.
Oral capsule formulation
[0231] The granulate from the previous section of this Example is filled into hard gelatin
capsules of a size suitable to the intended dose. The capsule is banded for sealing,
if desired.
Softgel formulation
[0232] A softgel formulation is prepared as follows:
|
% w/w |
Compound of this invention |
20.0 |
Polyethylene glycol 400 |
80.0 |
[0233] The compound is dissolved or dispersed in the polyethylene glycol, and a thickening
agent added if required. A quantity of the formulation sufficient to provide the desired
dose of the compound is then filled into softgels.
Parenteral formulation
[0234] A parenteral formulation is prepared as follows:
|
% w/w |
Compound of this invention |
1.0 |
Normal saline |
99.0 |
[0235] The compound is dissolved in the saline, and the resulting solution is sterilized
and filled into vials, ampoules, and prefilled syringes, as appropriate.
Controlled-release oral formulation
[0237] One Kg of a compound of this invention is coated in a modified Uni-Glatt powder coater
with Dow Type 10 ethyl cellulose. The spraying solution is an 8% solution of the ethyl
cellulose in 90% acetone to 10% ethanol. Castor oil is added as plasticizer in an
amount equal to 20% of the ethyl cellulose present The spraying conditions are as
follows: 1) speed, 1 liter/hour; 2) flap, 10-15%; 3) inlet temperature, 50°C, 4) outlet
temperature, 30°C, 5) percent of coating, 17%. The coated compound is sieved to particle
sizes between 74 and 210 microns. Attention is paid to ensure a good mix of particles
of different sizes within that range. Four hundred mg of the coated particles are
mixed with 100 mg of starch and the mixture is compressed in a hand press to 1.5 tons
to produce a 500 mg controlled release tablet.